What is Microbiology?

Microbiology, study of microorganisms, or microbes, a diverse group of generally minute simple life-
forms that include bacteria, archaea, algae, fungi, protozoa, and viruses. The field is concerned with
the structure, function, and classification of such organisms and with ways of both exploiting and
controlling their activities.
Microscope:

Microscope, is an instrument that produces enlarged images of small objects, allowing the observer
an exceedingly close view of minute structures at a scale convenient for examination and analysis in
the laboratory.
Types of Laboratory Microscopes:
There are many different types of microscopes used in modern pathology laboratories and research
departments around the world.
These typically include stereo, compound, digital and pocket microscopes, as well as electron, and
fluorescence microscopes.
How Does Light Microscopy Work?
As the name suggests light is the principal behind these types of microscopes, and the size of the
image seen is determined by the angle of light entering the eye. Therefore a glass lens is used as it
slows the light causing the wavelength of the light to become shorter and as a result light bends
(refraction). The amount bent is called the refractive index. The lens within the light microscope
serves to focus light rays at a specific place called the focal point. The focal length is the distance
between the center of the lens and the focal point. The strength of the lens is related to focal length as
the shorter the focal length, the greater the magnification. Normal light microscopy is called bright
field, however, there are also specialist types of light microscopy methods called dark-field
microscopy, phase-contrast microscopy, polarised light microscopy as well as fluorescence and stereo
microscopy.

Types of Light Microscopes:

The most common light microscopes used in laboratories and research laboratories are the compound
microscopes and the stereo microscopes. The fluorescence microscope is also a type of light
microscope that uses a more intense light source and special filters.
The Compound Microscope
The compound microscope is a light microscope that utilizes photons (light) and lenses to magnify the
object under observation. It differs from other types of microscopes as it utilizes multiple lenses and
can achieve a high magnification typically reaching 1000x magnification. The lenses are called the
eyepiece lens and the objective lens. Compound microscopes have many uses in modern laboratories,
for example in pathology departments they are commonly used by physicians to observe patient
specimens e.g. tissues and cells and to look for cellular changes that could help to diagnose or screen
for diseases such as cancer.
The Stereo Microscope
A stereo microscope is another common microscope found in laboratories, yet due to its low
magnification has limited use. Stereo microscopes are typically below x100 magnification, yet they
allow specimens to be viewed in three dimensions. They are commonly used in tissue inspection and
microsurgery.
Fluorescent Microscopy
The fluorescence microscope utilizes fluorescence and phosphorescence to observe and study various
molecules of interest. The specimen absorbs the light then re-emits it with a longer wavelength, this is
usually achieved by attaching fluorochromes to the tissue or biomolecule of interest. A fluorochrome
or fluorophore is a fluorescent chemical that can re-emit light when the light is “excited”. This results
in biomolecules that can easily be observed and tracked under the microscope, this is commonly used
to confirm the presence of certain proteins e.g. growth factors important in the treatment of particular
types of cancers.
The Electron Microscope is Used in Many Laboratories:
The electron microscope is an extremely powerful microscope capable of magnifications of
x1,000,000 and resolutions of about 2 nm. They utilize the same principles as the light microscope,
yet instead of a light source, a beam of electrons is used. In laboratories, they are used by highly
trained professionals to investigate and observe various markers of cell differentiation to identify
tumors types, and in renal disease, to monitor disease progress. They also have a critical role in the
diagnosis of renal disease and a range of other conditions. Additionally, they are often used for
microorganism identification.

Parts of a Microscope:
There are three structural parts of the microscope i.e. head, arm, and base.
1. Head – The head is a cylindrical metallic tube that holds the eyepiece lens at one end and
connects to the nose piece at other end. It is also called a body tube or eyepiece tube. It
connects the eyepiece lens to the objective lens. The light coming from objectives will bend
inside this tube. In binocular microscopes, they are adjustable so that the viewer can adjust the
eyepiece for maximum visualization.
2. Arm – This is the part connecting the base to the head and the eyepiece tube to the base of the
microscope. It supports the head of the microscope and is also used when carrying the
microscope. Some high-quality microscopes have an articulated arm with more than one
joint, allowing more movement of the microscopic head for better viewing.
3. Base – The base is the lowermost part of the microscope that supports the entire microscope
structure. It provides stability for the microscope. Illuminators, light switches, and electrical
wiring systems are fitted in the base.

Optical parts of a microscope and their functions:

The optical parts of the microscope are used to view, magnify, and produce an image from a
specimen placed on a slide. These parts include:
1. Eyepiece – The eyepiece (ocular Lens) is closest to the viewer’s eye. They are located at the
top of the microscope. This part is used to look at the specimen. These lenses come in
different magnification powers from 5X to 30X, but the most common ocular lenses are of
10X or 15X magnification. They magnify the image for the second time.
2. Eyepiece tube – It’s the eyepiece holder. It carries the eyepiece just above the objective lens.
In some microscopes, such as the binoculars, the eyepiece tube is flexible and can be rotated
 for maximum visualization for variance in distance. For monocular microscopes, they are
none flexible.
3. Diopter Adjustment – Diopter Adjustment is a control knob present only in the binocular
microscope that is used to change focus on one eyepiece. It is used to correct any difference in
vision and compensate for the differences in vision between the viewer’s two eyes.
4. Nose piece – A nose piece is a movable circular structure that houses all the objective lenses.
It is also called the revolving turret. It is connected to the body tube and lies just above the
stage. It can be rotated clockwise or counterclockwise to increase or decrease the
magnification. The change in magnification results due to a change in the objective lens.
5. Objective lenses – The objective lens is the lens that is closest to the specimen. They are
fitted on the nosepiece. A standard microscope has 3 to 4 objective lenses of different
magnifying powers: 4X, 10X, 40X, and 100X. The objective lenses first receive the light
transmitted from the specimen and magnify the image for the first time. Objective lenses are
color-coded and are of different sizes. Size and color depend on the power of the lens. The
smallest lens is of the lowest power, and gradually, the longest will be of the highest power.
The high-power lenses i.e. 40X and 100X, are retractable, i.e., their end can be pushed
inward. In most optical microscopes, objective lenses with 100X or more magnification are of
oil immersion type.
6. The Adjustment knobs – Adjustment Knobs are the control knobs used to focus the
microscope on the specimen. These knobs are of two types;
a. Fine Adjustment Knob: Fine Adjustment Knob is used for fine adjustment. It is a smaller
knob and is used to move the stage up or down very slowly. The stage covers a very small
distance on each rotation of the fine adjustment knob. It is used to sharpen the image. It is
mostly used while viewing under high power.
b. Coarse Adjustment Knob: Coarse Adjustment Knob is used for focusing the image under
low power magnification. It is a larger knob and is used to move the stage up or down very
rapidly. The stage is raised or lowered rapidly with the help of a coarse adjustment knob.
7. Stage – This is the section in which the specimen is placed for viewing. They have stage clips
that hold the specimen slides in place. The most common stage is the mechanical stage, which
allows the control of the slides by moving the slides using the mechanical knobs on the stage
instead of moving them manually.
8. Stage Control Knobs – Stage Control Knobs are the control knobs used to move the stage
mechanically. There are two knobs; one for moving left and right and the other for moving
forward and backward. This will move the slide in the field of vision.
9. Aperture – This is a hole in the microscope stage through which the transmitted light from
the source reaches the stage.
10. Microscopic illuminator – A microscopic illuminator is a light source. In some compound
microscopes, a mirror, which reflects the light from an external source to the sample, is used.
In other optical microscopes, different electric bulbs of low voltages are used as a constant
light source. Commonly used illuminators are tungsten-halogen lamps, 75-150W Xenon
lamps, tin-halide lamps, mercury vapor lamps, etc. The selection of types of bulbs is based on
the requirement of intensity and wavelength for illumination.
11. Condenser – These are lenses that are used to collect and focus light from the illuminator into
the specimen. They are found under the stage next to the diaphragm of the microscope. They
play a major role in ensuring clear, sharp images are produced with a high magnification of
 400X and above. The higher the magnification of the condenser, the clearer the image. More
sophisticated microscopes come with an Abbe condenser that has a high magnification of
about 1000X.
12. Diaphragm – It’s also known as the iris. It is found under the stage of the microscope, and its
primary role is to control the amount of light that reaches the specimen. It’s an adjustable
apparatus, hence controlling the light intensity and the size of the beam of light that gets to
the specimen. For high-quality microscopes, the diaphragm comes attached with an Abbe
condenser, and combined, they are able to control the light focus and light intensity that
reaches the specimen.
13. Condenser focus knob – This is a knob that moves the condenser up or down, thus
controlling the focus of light on the specimen.
14. Abbe Condenser – This condenser specially designed for high-quality microscopes makes
the condenser movable and allows very high magnification above 400X. High-quality
microscopes normally have a higher numerical aperture than objective lenses.
15. The rack stop – It controls how far the stages should go, preventing the objective lens from
getting too close to the specimen slide, which may damage the specimen. It is responsible for
preventing the specimen slide from coming too far up and hitting the objective lens.
16. Light Switch – Light Switch is an electrical control device. Light switches are used to on and
off the illuminator.
17. Brightness Adjustment – The brightness adjustment system controls the voltage supplied to
the light bulb, controlling the intensity (brightness) of the light bulb.

Sterilization:
Sterilization is the complete eradication of microorganisms (fungi, bacteria, and viruses) present on
the surface of any material. The microorganisms can be removed or destroyed from the definite
materials (seeds, leaves, root pieces, stem pieces) by using sterilization techniques.

Principle of Sterilization Techniques

Maintaining a sterile environment

during the transfer, or culturing of
cells or tissues of microbes is most
important.

The concept of sterilization, for

making the materials free from any
type of contamination was given
by Louis Pasteur. Thus sterilization is
a process of making an article,
surface, or medium free from any
type of microorganisms that
contaminate the object and provide
unwanted results.

Some other methods used to make these sterile are disinfection and incineration.
• Disinfection: Disinfection is a similar process of killing harmful organisms, especially the
objects but not the culture media. Disinfection of tabletops, equipment, and other surfaces is
usually done by using glycolic acid compounds, carbolic acid, formaldehyde, ethanol, etc.
• Incineration: It is a process of killing microorganisms by using a flame, therefore, it is called
flame sterilization. It is done by keeping the inoculation needle over the flame of the Bunsen
burner till it becomes red hot. Thus, the microorganisms present on the surface of the needle
are destroyed.

Different methods of sterilization:

Physical Methods of Sterilization:
There are several physical methods of sterilization of materials and objects. These are the
following:
Moist Heat
Culture media (liquid and agar), water, glassware, surgical blades, and scalpels are sterilized by
using moist heat (steam under pressure). It is done by using an autoclave, and also by a pressure
cooker (used for cooking pressure at home).
Dry Heat
Dry heat sterilization is carried out by using a hot air oven. Glassware, glass syringe, forceps,
scalpel, pipettes, flasks metallic instruments, Petri dishes, etc. are sterilization in an oven at 150°C
for 1 hour or 250°C for 30 minutes.
Radiation
Normally UV radiation is used in an inoculation chamber or laminar airflow. Expose the working
area to UV radiation before 2 hours to start the work. The source of UV radiation is UV lamps or
tubes enclosed in quartz because the glass will not transmit UV radiation.
Care should be taken not to see the UV radiation with naked eyes. Otherwise, any abnormality
may occur in the eyes.
Membrane Filtration
Sterilization of heat-sensitive substances like enzymes, antibiotics, and amino acids could not be
done by autoclaving because these may be denatured and rendered non-functional.
Hence, these are sterilization through various types of filters which may retain bacteria.

Chemical Methods of Sterilization:

There are several chemical methods of sterilization of materials and objects. These are the
following:
Alcohol
Ethanol (70%) or propane (70%) is used to sterilize the working tabletop, inoculation chamber,
hands, materials to be used, glass apparatus, etc.
Aldehyde
Generally, the laboratory or chamber is fumigated with formaldehyde when the number of
contaminants gets increases.
 Inorganic Chemicals
There are certain chemicals toxic to any organisms such as salts of copper, mercury, etc.
HgCl2 solution (0.1%) is most commonly used as a disinfectant for seeds, explants, and plant
materials. For the same purpose, other chemicals used are sodium hypochlorite (NaOCl) (10%).
The materials to be disinfected in the solution are kept in HgCl2 solution for 5-10 minutes (for
naked hypochlorite). Soon take out the materials, transfer them into sterilized distilled water, and
washed properly. Again repeat the process of washing 5-8 times to remove the traces of
chemicals.

Requirements of Sterilization Techniques:

• Autoclave (for moist heat sterilization), laminar airflow (fitted with UV tube)
• Oven (for dry heat sterilization)
• Seed/leaves/root pieces/stem pieces of any plants.
• Spirit, Ethyl alcohol (70%), Mercuric chloride (0.1% HgCl2), Sodium
• hypochlorite/Calcium hypochlorite (10%) (for chemical sterilization)
• Vacuum pump, filtration unit, and Whitman filter paper (0.22 μm) (membrane filtration
sterilization)
• Sterile distilled water, glassware, cotton plugs, Bunsen burner, absorbent cotton, plasticware,
Petri dishes, etc.
• Surgical blades, holders, forceps, etc.

Procedures of Sterilization Techniques:

Sterilization of Glassware and Objects:

For complete sterilization suitable containers having glassware, glass syringe, forceps, surgical blades
holder, needles, scalpels, and metal instruments are kept in an oven at 150°C for 1 hour or 250 °C for
30 minutes.

Sterilization of Nutrient Media:

Culture media (broth or agar media), water, and other material (Millipore filter) are sterilized by an
autoclave (high-pressure stem using moist heat) at 15 psi (pound per inch square) for 20-30 minutes
which gives 121°C.

Glassware must be wrapped with aluminum foil and flasks containing nutrient medium, must be
plugged with cotton, and then wrapped with aluminum foil. After sterilization, the materials should
not be taken out immediately.
Then wait for 15-20 minutes to lower the temperature to about 60°C. After releasing the pressure, the
material should be taken out.

Surface Sterilization:

For surface sterilization of seeds and other parts of a plant (leaves, roots, bark), these are washed with
tap water. Then it is dipped in disinfectant chemical solutions first in 70% Ethyl alcohol for 30
seconds, then in 10% sodium hypochlorite for about 10-20 minutes.
The time of sterilization varies with the materials used. The materials should be thoroughly washed
(5-10 times) with double distilled water (5-8 times repeat this process). Sterilization procedures
should be performed under aseptic conditions (laminar airflow).
Sometimes seeds are dipped only in 10% Sodium hypochlorite or Calcium hypochlorite for 5-10
minutes of sterilization without any other treatment.

Membrane Filtration Method:

The membrane filtration method is used only for those chemicals which are heat sensitive or unstable
at high temperatures (enzymes, antibiotics, vitamins, amino acids).
Commonly, the Whatman filter (0.22 μm) is used for sterilization. These substances are passed
through ultra-filtration membrane filters using a vacuum pump.

Precautions of Sterilization Techniques:

• Before the operation of laminar flow, the working surface should be sterilized with 70% Ethyl
alcohol and UV radiation for 30 minutes.
• Hands should be washed with 70% Ethyl alcohol before handling the laboratory materials.
• Surgical hand gloves should also be used while performing the experiments under aseptic
conditions.

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 use of moist heat facilitates the killing of all microorganisms, including heat-resistant
endospores which is achieved by heating the materials inside the device at temperatures above the
boiling point of water. According to the principle of gas laws, this can be achieved by raising
the pressure inside the device.
The usual procedure is to heat at 1.1 kilograms/square centimeter (kg/cm2) [15 pounds/square
inch (lb/in2)] steam pressure, which yields a temperature of 121°C. At 121°C, the time of
autoclaving to achieve sterilization is generally considered to be 15-20 min, depending on the
volume of the load.

Procedure:
1. Place the material to be sterilized inside the pressure chamber and fill the cylinder with
sufficient water
2. Close the lid and put on the electrical heater.
3. Adjust the safety valve to the required pressure.
4. After the water boils, allow the steam and air mixture to escape through the discharge tap till
all the air has been displaced
This can be tested by passing the steam-air mixture liberated from the discharge tap into a pail of
water through a connecting rubber tube. When the air bubbles stop coming in the pail, it indicates
that all the air has been displaced by steam.
5. Close the discharge tap.
The steam pressure rises inside and when it reaches the desired set level (e.g. 15 pounds (lbs) per
square inch in most cases), the safety valve opens and excess steam escapes out.
6. Count the holding period from this point of time, which is about 15 minutes in most cases.
7. After the holding period, stop the electrical heater and allow the autoclave to cool until the
pressure gauge indicates that the pressure inside is equal to the atmospheric pressure.
8. Open the discharge tap slowly and allow the air to enter the autoclave.

9. Open the lid of the autoclave and remove the sterilized materials.

Laminar Air Flow: A Laminar flow hood/cabinet is an enclosed workstation that is used to create a
contamination-free work environment through filters to capture all the particles entering the cabinet.
• These cabinets are designed to protect the work from the environment and are most useful for
the aseptic distribution of specific media and plate pouring.
• Laminar flow cabinets are similar to biosafety cabinets with the only difference being that in
laminar flow cabinets the effluent air is drawn into the face of the user.
• In a biosafety cabinet, both the sample and user are protected while in the laminar flow
cabinet, only the sample is protected and not the user.

Working Principle of Laminar Airflow Chamber:

• The operation of the laminar airflow chamber is based on the unidirectional flow of
filtered sterile air with constant velocity. The prefilter/filter pad of the hood traps the
outside non-sterile air; the air is slightly filtered by it. The prefilter air is then blown
to the HEPA filter with the help of a fan/blower. The HEPA filter is a highly efficient
filter capable of trapping air pollutants with a size of 0.3 µ or larger. Now, the
completely sterile air passes through the working area of the hood. This way, the non-
sterile air is trapped, filtered, and sterilized to pass through the working area.
• Sometimes the objects used inside the hood might be non-sterile or exposed to the
outside air. The exposure may risk the contamination of the hood, so using the UV
 lamp while using metals, glassware, and media before performing the task helps
maintain sterility.

Incubator: An incubator, in microbiology, is an insulated and enclosed device that provides an

optimal condition of temperature, humidity, and other environmental conditions required for the
growth of organisms.
An incubator is a piece of vital laboratory equipment necessary for cultivating microorganisms under
artificial conditions.
An incubator can be used to cultivate both unicellular and multicellular organisms.
Principle/ Working of Incubator:
• An incubator is based on the principle that microorganisms require a particular set of
parameters for their growth and development.
• All incubators are based on the concept that when organisms are provided with the optimal
condition of temperature, humidity, oxygen, and carbon dioxide levels, they grow and divide
to form more organisms.
• In an incubator, the thermostat maintains a constant temperature that can be read from the
outside via the thermometer.
• The temperature is maintained by utilizing the heating and no-heating cycles.
• During the heating cycle, the thermostat heats the incubator, and during the no-heating period,
the heating is stopped, and the incubator is cooled by radiating heat to the surrounding.
• Insulation from the outside creates an isolated condition inside the cabinet, which allows the
microbes to grow effectively.
• Similarly, other parameters like humidity and airflow are also maintained through different
mechanisms that create an environment similar to the natural environment of the organisms.
• Similarly, they are provided with adjustments for maintaining the concentration of CO2 to
balance the pH and humidity required for the growth of the organisms.
• Variation of the incubator like a shaking incubator is also available, which allows for the
continuous movement of the culture required for cell aeration and solubility studies.

Classifications of Microorganisms:- The major groups of microorganisms—namely

bacteria, archaea, fungi (yeasts and molds), algae, protozoa, and viruses.

• Bacteria (eubacteria and archaea): All bacteria are prokaryotic—that is, single-celled
organisms without a membrane-bound nucleus. Their DNA (the genetic material of the cell),
instead of being contained in the nucleus, exists as a long, folded thread with no specific
location within the cell.
• Algae: Unlike bacteria, algae are eukaryotes and, like plants, contain the green
pigment chlorophyll, carry out photosynthesis, and have rigid cell walls. They normally occur
in moist soil and aquatic environments. These eukaryotes may be unicellular and microscopic
in size or multicellular and up to 120 metres (nearly 400 feet) in length.
• Fungi: Fungi are eukaryotic organisms that, like algae, have rigid cell walls and may be
either unicellular or multicellular. Some may be microscopic in size, while others form much
larger structures, such as mushrooms and bracket fungi that grow in soil or on damp logs.
• Protozoa: Protozoa, or protozoans, are single-celled, eukaryotic microorganisms. Some
protozoa are oval or spherical, others elongated. Still others have different shapes at different
stages of the life cycle. Cells can be as small as 1 μm in diameter and as large as 2,000 μm, or
2 mm (visible without magnification).
• Viruses: Viruses, agents considered on the borderline of living organisms, are also included
in the science of microbiology, come in several shapes, and are widely distributed in nature,
infecting animal cells, plant cells, and microorganisms.
• Prions: Even smaller than viruses, prions (pronounced “pree-ons”) are the simplest infectious
agents. Like viruses they are obligate parasites, but they possess no genetic material.
 Although prions are merely self-perpetuating proteins, they have been implicated as the cause
of various diseases, including bovine spongiform encephalopathy (mad cow disease), and are
suspected of playing a role in a number of other disorders.
• Lichens: Lichens represent a form of symbiosis, namely, an association of two different
organisms wherein each benefits. A lichen consists of a photosynthetic microbe (an alga or a
cyanobacterium) growing in an intimate association with a fungus. A simple lichen is made up
of a top layer consisting of a tightly woven fungal mycelium, a middle layer where the
photosynthetic microbe lives, and a bottom layer of mycelium.
• Slime molds: The slime molds are a biological and taxonomic enigma because they are
neither typical fungi nor typical protozoa. During one of their growth stages, they are
protozoa-like because they lack cell walls, have amoeboid movement, and ingest particulate
nutrients. During their propagative stage they form fruiting bodies and sporangia, which bear
walled spores like typical fungi.
 Structure of Bacteria

Bacteria is a unicellular prokaryotic organism. The structure of the bacteria consists of three major
parts: Outer layer (cell envelope), cell interior, and additional structures.
• Outer layer (Cell envelope): It includes the cell wall of bacteria and the plasma
membrane beneath it. The outer envelope acts as a structural and physiological barrier
protecting the interior of the bacterial cell from the outer external environment. The function
of the cell envelope is to protect the bacteria from the osmotic lysis and give the bacteria its
shape.
• Cell interior: The internal structure of the bacterial cell consists of the protoplasm, which
consists of the cytoplasm, cytoplasmic inclusions (mesosome, ribosomes, inclusion granules),
and single circular DNA.
• Additional structures: It includes capsule, flagella, fimbriae, and spores.

Cell wall:
 The bacteria’s cell wall is the outer rigid and chemically complex structure. It is in between the cell membrane
and the capsule/slime layer. The cell wall of the bacteria maintains the shape of the cell and protects the bacteria
from changes in osmotic pressure. The bacteria’s cell wall makes up 20-30% of the cell’s dry weight. The major
component of the cell wall is the peptidoglycan layer. Peptidoglycan is the disaccharide and consists of the N-
acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) as the sugar derivatives. Based on the structure
of the cell wall, bacteria are classified into Gram-positive and Gram-negative bacteria.

Differences between Gram-positive cell wall and Gram-negative cell wall

Gram-positive cell wall Gram-negative cell wall

Gram-positive cell wall is thick, i.e., 15-80 nm. Gram-negative cell wall is thin, i.e., 2 nm.

Peptidoglycan layer is present in abundance. Peptidoglycan layer is significantly less.

Lipid content is 2-5%. Lipid content is 15-20%.

Teichoic acid is present. Teichoic acid is absent.

Treatment with lysozyme makes protoplast. Treatment with lysozyme makes spheroplast.

Gram-positive cell wall:

Peptidoglycan is the main component of the Gram-positive cell wall that is thick and constitutes about
40-80 % of the dry weight of the cell wall. The Gram-positive cell wall consists of Teichoic acids and
Teichuronic acid.
Gram-negative cell wall:
Gram-negative cell wall contains four main components: lipopolysachharides, outer membrane,
lipoprotein layer, and peptidoglycan.
Lipopolysaccharides
LPS is present in the outer membrane of the Gram-negative bacteria. It comprises lipid A, core
oligosaccharide, and O polysaccharide.
Outer membrane:
The outer membrane in the Gram-negative bacteria protects the cell from various environmental stress
and harmful components. It prevents the cell from the entry of the antibiotics and also protects the cell
lysis from the lysozyme. The outer membrane of Gram-negative bacteria is the bilayered structure,
and it consists of the following parts: porins, outer membrane proteins (OMPs), and other proteins.
• Porins: Porin is present in large amounts in the outer membrane. Its main role is in
maintaining outer membrane permeability and allowing the passive diffusion of low
molecular weight hydrophilic substances like sugars, amino acids, and certain ions.
• Outer membrane proteins (OMPs): Outer membrane proteins (OMPs) are embedded in the
outer membrane. They are involved in nutrient uptake, membrane homeostasis, and virulence.
The four major proteins involved in transmembrane diffusion of maltose and maltodextrins
 are Omp C, D, F, and PhoE & LamB. Omp A protein anchors the outer membrane to the
peptidoglycan layer.
• Lipoprotein layer:
Lipoprotein stabilizes the outer membrane of the Gram-negative cell wall and is composed of
the Braun lipoprotein.

Peptidoglycan:
Peptidoglycan layer in the Gram-negative bacteria is less than that of the Gram-positive bacteria. It is
2-3 nm thin and is bound by the lipoprotein and the plasma membrane.
Periplasmic space:
The space between the cell membrane and the outer membrane in Gram-negative bacteria is called
periplasmic space. It consists of different enzymes and proteins. E.g., hydrolytic enzymes and beta-
lactamase binding proteins.
Acid-fast bacilli cell wall:
The cell of acid-fast bacteria is resistant to most detergents and strong acids. It cannot be stained with
the Gram stain, so we need to perform the Ziehl-Neelsen staining for its visualization. Acid-fast
bacteria like Mycobacterium tuberculosis consist of mycolic acids linked to the arabinoglycan protein.
Due to its structural composition, it resists the acid alcohol, called the acid-fast bacilli.
Cell wall deficient forms:
There are many types of bacteria which is devoid of the cell wall. The different cell wall deficient
forms of bacteria are protoplast, spheroplast, Mycoplasma, and L-forms.

When Gram-positive bacteria are treated with lysozyme, protoplast is formed. In the protoplast, the c
Protoplast
absent, but its cytoplasmic membrane is intact.

Spheroplast When Gram-negative bacteria are treated with lysozyme, spheroplast is formed.

Mycoplasma Mycoplasma is a naturally occurring bacteria in which a cell wall is absent.

L-form is named after the Lister Institute, London, where it was first demonstrated in the Streptobaci
L-forms
moniliformis.

Plasma membrane or cytoplasmic membrane:

A plasma membrane or cytoplasmic membrane is a thin semipermeable membrane lying beneath the
cell wall. Sterol-like cholesterol is absent in the bacterial cell membrane except
in Mycoplasma, which makes it different from the eukaryotic cell membrane. The cytoplasmic
membrane is a barrier and controls the metabolites’ inflow and outflow. It also helps in electron
transport and oxidative phosphorylation.
Cytoplasm
The cytoplasm of the bacterial cell contains the ribosomes, mesosomes, and intracytoplasmic
inclusion bodies.
Ribosomes
The ribosome of bacteria is the 70S which is made up of 30S and 50S subunits. Ribosomes serve as
the site for protein synthesis.
Mesosomes
Mesosomes are analogs of the mitochondria present in eukaryotes. It is the main site of the respiratory
enzymes in bacteria.
Intracytoplasmic inclusion bodies:
Inclusion bodies are involved in storage, acting as a source of carbon, inorganic substances, energy,
and reduction of the osmotic pressure. Examples of intracytoplasmic inclusion bodies: are
metachromatic granules or volutin granules, starch inclusions, and lipid inclusions.
DNA
The bacterial cell contains circular double-stranded DNA. All the genetic information is encoded in
the DNA.
Plasmid
Plasmids are the extrachromosomal DNA that can replicate on its own. Its size ranges from 1.5
kilobases (kb) pairs to 120 kb pairs.
Capsule:
The capsule is the thin outer layer present in some bacteria. It protects the bacteria from phagocytosis.
Non-capsulated bacteria are more prone to phagocytosis than capsulated bacteria. By performing the
capsule staining, it can be visualized by the light microscope in which the capsule is seen as a hollow
structure.
Examples of the capsulated bacteria:
• Streptococcus pneumoniae
• Haemophilus influenzae
• Klebsiella pneumoniae
• Neisseria meningitidis
Flagella:
Bacterial Flagella help in locomotion. It protrudes out from the cell wall. Flagella are made up of
flagellin protein. Some bacteria in which flagella is absent are called atrichous bacteria. The bacteria
in which flagella are present are termed the flagellated bacteria. Based on the number and the position
of flagella, bacteria are classified into four types.
• Monotrichous: one flagellum present on one side of the body. i.e., polar flagella
• Lophotrichous: a cluster of the flagella is present on one end of the body
• Amphitrichous: either one or cluster of flagella is present on both ends of the body
• Peritrichous: flagella present all over the body
Pili (Fimbriae):
These are the hair-like filaments extending from the cell surface. Pili are shorter and straighter than
the flagella. It is made up of pilins protein. Pili helps in the adherence of the bacteria to the host cells.
Sex pili help in the transfer of bacterial DNA during conjugation.
Spores:
Spores are the resting or dormant form of bacteria. During starvation or under unfavourable
conditions, bacteria like Bacillus and Clostridium spp. produces spores. The spores of the bacteria are
resistant to boiling, disinfectants, and heating. Bacterial spores can be visualized by endospore
staining. Examples of the spore former bacteria: Bacillus stearothermophilus, Bacillus anthracis,
and Clostridium tetani.

Different Shapes of Bacteria:

➢ Cocci:
1. The bacteria that are oval or spherical in shape are known as cocci bacteria
2. These can be solitary or connected to one another in a group. When grouped
together, they seem flattened.
3. Coccoid forms are thought to have evolved from rod-shaped creatures
throughout evolutionary time.

➢ Arrangements of Cocci:
1. Cocci bacteria can be organised individually, in pairs, in four-cell groups, in chains,
in clusters, or in eight-cell cubes.
2. During cell division, these cells stay together. The bacteria’s form is altered by the
plane of cell division.
3. Cocci have a gram-positive cell wall with a thick peptidoglycan layer or a gram-
negative cell wall with a thin peptidoglycan layer.
d. According to the arrangement of cells, the cocci are again divided into the
following subtypes:
A. Monococcus – It is a bacterial species that consists of a single cell.
B. Diplococcus
i. When two bacterial cells form a pair, this configuration occurs (joined together).
ii. Some cells in this arrangement may be spherical, while others may be flattened, elongated,
or bean-shaped.
iii. Examples: Streptococcus pneumoniae.
C. Streptococcus
i. In this type, the bacteria are organised in long chains here.
ii. These bacteria belong to the Streptococcaceae family, which is characterised by Gram-
positive bacteria and a lack of motility.
iii. Streptococcus pneumonia, Streptococcus pyogenes, and Streptococcus mutans are some
examples.
D. Tetrads
i. Tetrad bacteria are organised in a group of four cells that remain connected during cell
division and development in the attachment.
ii. When the cells split into two planes, this pattern occurs.
iii. Aerococcus, Pediococcus, and Tetragenococcus are some examples.
E. Staphylococcus
i. Bacteria organised in grape-like clusters make up this kind of arrangement.
ii. This is caused by cell division in both planes and is characterised by immotile and Gram-
positive organisms.
iii. Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus aureus, and other
bacteria are examples.
F. Sarcinae
i. The bacterial cells form an eight-cell cluster in this configuration.
ii. This happens when the cells divide in a perpendicular plane.
iii. The fact that these organisms are strictly anaerobic in nature.
iv. Sarcina lutea, Sarcina aurantiaca, and Sarcina ventriculi are other examples.

➢ Bacilli (Rod-shaped)
These are rod-shaped cells that, like cocci, can exist alone or in association with other cells. Bacilli
bacteria were among the earliest to emerge, and their form is considered to be less favourable than
that of other bacteria.

• Arrangement of Bacilli
1. Bacillus
i. Bacilli are bacteria that are rod-shaped and exist as solitary cells.
ii. These bacteria are facultative anaerobes that may generate endospores
iii.Salmonella enterica subsp., Bacillus cereus and Salmonella choleraesuis are some examples.
2. Diplobacilli
i. Diplobacilli, like Diplococci, is found in pairs.
ii. The two cells do not divide and develop in an associated configuration after cell division.
iii. Coxiella burnetii, Klebsiella rhinoscleromatis, and Moraxella bovis are some examples.
3. Streptobacilli
i. Bacteria in this category are arranged in chains.
ii. This happens when a single chain of cells divides.
iii. Streptobacillus moniliformis, Streptobacillus felis, Streptobacillus Levaditi, and Streptobacillus
hongkongensis are examples of streptobacilli.
4. Coccobacilli
i. As the name indicates, Coccobacilli is similar to both cocci and bacilli.
ii. Because they are smaller, they look stumpy.
iii. Chlamydia trachomatis, Gardnerella vaginalis, and Haemophilus influenzae are some of the
examples.
5. Palisades
i. Palisades are bacilli bacteria that have a picket fence-like shape due to a bend at the site of division
during cell division.
ii. They have the appearance of Chinese characters.
iii. Example – Corynebacterium diphtheria.
 ➢ Spiral
This group of microorganisms includes bacteria that are either helical-shaped or curved or comma-
shaped. The bacterium might have a corkscrew-like spiral or be slightly bent.

1. Vibrio
i. These are the comma-shaped bacteria that are slightly bent.
ii. Vibrio mytili, Vibrio anguillarum, Vibrio parahaemolyticus, and Vibrio cholera are some examples.

2. Spirochetes
i. Spirochetes are spiral bacteria that have a helical shape.
ii. These organisms are flexible and have an axial filament which helps in motility. These filaments
are a key feature that distinguishes spirochetes from other bacteria.
iii. These filaments travel the length of the bacterium, aiding in the twisting of the bacteria’s motility.
iv. Examples include Leptospira interrogans, Treponema pallidum, etc.

3. Spirilla (Helical-shaped/Corkscrew form)

i. Spirochetes have a similar structure to these bacteria, although they are more rigid.
ii. They, too, have a flagellum, but unlike spirochetes, they lack the endoflagella.
iii. Helicobacter pylori, Campylobacter jejuni, and Spirillum winogradskyi are some of the examples.

➢ Other Shapes and Arrangements

Some of the other shapes and arrangements are as follows:
1. Filamentous Bacteria
i. These are filament-shaped bacteria that are thin, long, and are filamentous.
ii. They can split into mycelium-like branches that look like strands of hair or spaghetti.
iii. Actinomycetes are a good example.
2. Appendaged Bacteria
i. Appendaged bacteria are bacteria that generate a distinct structure, such as pillus or fimbriae.
ii. These bacteria are more virulent when compared to the other bacteria that do not form these
appendages.
iii. Example – Neisseria gonorrhoea.
3. Club-shaped Rod Bacteria
i. One side of these bacteria is thinner than the other.
ii. Corynebacterium is a well-known representative of this group.
4. Box-shaped/ Rectangular Bacteria
i. Bacteria with a box-like form or that are rectangular in shape.
ii. Box-shaped bacteria have a rectangular form that resembles the shape of a box.
iii. Example – Haloarcula marismortui.
5. Triangular-shaped Bacteria
i. This group includes bacteria that are triangular in shape.
ii. Example: Haloarcula.
6. Pleomorphic Bacteria
i. The bacteria that belong to this group does not have or possess a specific shape.
ii. They can alter the shape, although they appear to have a distinct morphology in pure cultivation.
iii. Examples: Mycoplasma.
7. Star-shaped Bacteria
i. The bacteria that look like stars or are star-shaped are included in this group of bacteria.
ii. Examples: Stella humosa.
8. Stalked Bacteria
i. These are the bacteria that possess a stalk on one end of the cell.
ii. Examples: Caulobacter crescentus.
 Bacterial Growth Factors

Bacterial growth factors primarily include.

1. Temperature
2. pH
3. Oxygen Concentration
4. Carbon Dioxide
5. Light
6. Osmotic Pressure

Temperature

It is one of the most crucial factors which decides the multiplication rate of microorganisms.
A temperature can be minimal, optimal and maximal.

• Minimal temperature: Below which no growth occurs.

• Optimal temperature: At which fastest growth occurs.
• Maximal temperature: Above which no growth occurs.
pH:

Bacteria are sensitive to various pH range. The bacterial species that grow at a pH range
above or below the preferred value would not survive. But, bacteria at optimum pH show the
best growth in nearly neutral pH, i.e. 6.5-7.5.

Oxygen Concentration
The bacterial species that use molecular oxygen (O2) produce more energy from nutrients
than anaerobes. Oxygen functions as a terminal electron acceptor for an electron transport
chain during aerobic respiration.

Light

It is another factor that affects the bacterial growth, and those bacteria which makes the use of
light source can be classified as:
• Phototrophs: It refers to a group of bacteria, which derives energy by capturing
photons mainly from the sunlight. Phototrophs can be either classified into autotrophs
(fix carbon) or heterotrophs (utilizes carbon). Examples: Rhodobacter
capsulatus, Chromatium, Chlorobium etc.
• Chemotrophs: It refers to the group of bacteria, which derives energy by oxidizing
electrons primarily from chemical sources. Organic (chemoorganotrophs) or
inorganic (chemolithotrophs) are the two common types. These predominate in the
ocean floors where the sunlight cannot reach.

Osmotic Pressure
Microbes require minerals or nutrients for their growth, which can be obtained from the
surrounding water. Osmotic pressure and salt concentration of the solution can influence
bacterial growth. The bacterial cell wall gives a mechanical strength that allows the bacteria
to withstand alternations in the osmotic pressure.
Osmophilic bacteria require high osmotic pressure. When the bacterial cell is subjected to the
hypertonic solution, it may cause osmotic water removal, resulting in plasmolysis or osmotic
shrinkage of the protoplasm.
In contrast, when the bacterial cell is subjected to the distilled water from the high
concentration, it may cause excessive water imbibition resulting in plasmoptysis or cell
bursting.

Binary Fission: Binary fission is the process through which asexual reproduction happens in
bacteria. During binary fission, a single organism becomes two independent organisms. Binary fission
also describes the duplication of organelles in eukaryotes. Mitochondria and other organelles must
reproduce via binary fission before mitosis so each cell has ample organelles.

• Process: Binary fission is a relatively simple process, compared to mitosis, because binary
fission does not involve reproducing organelles or complex chromosomes. The process starts
with the replication of the DNA within the cell. Mitochondria must also replicate
their DNA before binary fission, though other organelles have no DNA.
Then, the DNA is separated into alternate ends of the single cell. The plasma membrane pinches the
cell apart, and one cell becomes two. With a fully-functioning DNA molecule, each cell is then
capable of all the functions of life. Therefore, the cells become independent organisms.
Organelles, though they are not independent organisms, separate in this way as well. Endosymbiotic
theory says that mitochondria and chloroplasts were once independent organisms that have evolved to
live within other cells. As such, they still replicate via binary fission.

Bacterial Growth Curve:

• The bacterial growth curve is a graphical representation that illustrates the different phases of
bacterial growth over time. It provides valuable insights into the growth patterns and
dynamics of bacterial populations.

• The curve typically consists of four distinct phases: lag phase, log phase (exponential growth
phase), stationary phase, and death phase. These phases reflect the changes in the number, size, and
mass of bacterial cells during their growth cycle.
• Several factors influence bacterial growth, including temperature, pH, oxygen availability, nutrient
availability, and moisture content. Bacteria are prokaryotic and unicellular organisms that primarily
reproduce through a process called binary fission, where one parent cell divides into two identical
daughter cells.
• In the log phase, bacterial growth is at its maximum, with the number of viable bacteria increasing
exponentially. This phase is of particular interest in microbiological research and various
applications. The stationary phase occurs when the rate of cell division equals the rate of cell death,
resulting in a stable population size. Finally, the death phase is characterized by a decline in the
number of viable bacteria, often due to nutrient depletion and accumulation of waste products.
 Stains
❖ Gram Staining or Crysal Violet staining:
Principle:
The basic principle behind this technique is that some bacteria have the ability to retain the dye while
the other bacteria don’t have the ability to retain the dye after staining with crystal violet dye. This
differentiation is possible due to the difference in the cell wall of bacteria.
1. Gram positive bacteria have high peptidoglycan content and two types of teichoic acids, wall
teichoic acid and lipoteichoic acid. Wall teichoic acid keeps the cell wall intact and
lipoteichoic acid have affinity toward anionic crystal violet dye. When we pour crystal violet
(first step), it gets attached to lipoteichoic acid and reaches to the cell cytoplasm where it bind
to magnesium ion and forms a CV-Mg-RNA complex, while in gram negative bacteria there
is no teichoic acid and have less content of peptidoglycan. So the dye did not penetrate so
well and forms a weaker complex.
2. In Second step, iodine is poured which act as a mordant i.e., it intensify the color and form a
stronger complex in gram positive bacteria which is known as CV-Mg-RNA-I complex.
3. In third step, ethanol is poured that has a dual property (1) lipid solvent and (2) dehrydation.
So lipid gram positive has low lipid content, which get easily dissolved and form small pores
which easily get shrink also that prevent washing off of complex in gram positive bacteria and
appear purple at this time and in Gram-negative bacteria have a high content of lipid this
results in formation of large pores which does not get shrink so easily. This causes to flood off
the complex from gram negative bacteria and appear colorless this time.
4. In fourth step, safranin is poured which stains the gram negative bacteria red in color.

Requirements:
1. Crystal violet (primary stain)
2. Iodine (mordant)
3. 95% Ethanol (decolorizer)
4. Safranin
5. Glass slide
6. Inoculating loop
7. Burner
Procedure:

1. Firstly, get a free slide which should be clean and free from grease.
2. With the loop pour the sample and make a smear of it.
3. Dry the slide with air and then provide heat to the slide. This heat is given to the sample to fix
the sample on the slide so that it doesn’t get washed off too easily. Moreover, through the
heat, some of the bacteria get killed but it gets fixed properly.
4. Now after the heat finally the dye (crystal violet) is poured onto the slide and minimum for 30
seconds and maximum for 1 minute it is kept on the slide and then it is washed with water.
This time is given so that the bond is formed between the dye and the cell membrane of the
bacteria. Moreover, through the heat, some of the bacteria get killed but it gets fixed properly.
5. The mordant or it can be said the iodine solution is applied and it is also kept for a maximum
1 minute and then the slide is washed with normal water. It gets trapped in the cell after
making a bond with the crystal dye.
6. Now it’s the turn for acetone or alcohol to be used on the slide and then after 10-20 seconds,
the slide is washed again with water. These two agents work as decolorizers.
7. The last step is the application of safranin for 1 minute and then again it is rinsed with water.
8. The slide undergoes blot and air dry and now it is ready for observation under the microscope.
Result:
If the bacteria are coloured pink or red that shows Gram-negative bacteria. Gram-negative
bacteria lose the stain.

Gram Negative Bacteria

Gram positive bacteria are those which keep primary stain i.e. crystal violet due to their thick
peptidoglycan layer in cell wall and appear purple in light microscope.

Gram Positive Bacteria

❖ Acid-Fast staining or Ziehl-Neelsen (ZN) Staining (Hot Method):

Microorganisms in the genus Mycobacteria and Nocardia have complex cell walls with higher
lipid content. The cell wall of these microbes contains mycolic acid, which is a complex
hydrocarbon. Due to this waxy cell wall, bacteria don’t take up the stain easily. Therefore,
microorganisms with a waxy cell wall are difficult to stain with routine staining methods such
as gram staining or simple staining. To visualize such bacteria special staining method such
as Ziehl Neelsen acid fast stain (ZN Stain) is very useful.
• Principle: Acid-fast mycobacteria contain mycolic acid in their outer membrane,
making the cells waxy and resistant to staining with aqueous based stains such as the
Gram stain. The primary stain, hot carbolfuchsin is applied to the cells, and heat and
phenol are used to allow the stain to penetrate into the waxy surface of acid-fast
microorganisms. The excess stain is removed with treatment by acid alcohol (ethanol
and hydrochloric acid). A secondary stain, methylene blue, is then applied to the cells.
• Reagents:
Primary Stain: 0.3% Carbol-fuchsin. Dissolve 50 g phenol in 100 mL ethanol (95%) or
methanol (95%). Dissolve 3 g Basic fuchsin in the mixture and add distilled water to
bring the volume to 1 L.
Decolorization Solution: Add 30 mL hydrochloric acid to 1 L of 95% denatured
alcohol. Cool and mix well before use. Alternate decolorizing reagent (without
alcohol): Slowly add 250 mL sulfuric acid (at least 95%) to 750 mL distilled water.
Cool and mix well before using.
Counterstain: 0.3% methylene blue. Dissolve 3 g methylene blue in 1 L distilled
water.
• Procedure:

1. Flood the smear with primary stain carbon fuchsin. Keep the slide on steam for
stain penetration. wait for 5 minutes (Heat the carbol fuschin first in a test tube then
flood it over smear).
2. Allow the slide to cool and wash with tap water.
3. Now flood the smear with decolorizer (20% Sulfuric Acid) for 10-15 seconds.
4. Wash the smear on the slide with tap water.
5. Now counterstain the smear with methylene blue For about 2 minutes.
6. Wash the smear with tap water
7. Blot dry the slide and examine under oil immersion.

Result Interpretation of Acid Fast Stain:

Acid fast: Bright red to intensive purple, Red, straight or slightly curved rods,
occurring singly or in small groups, may appear beaded
Non-acid fast: Blue color; In addition, background material should stain blue.
 Acid-Fast Bacilli present

** ZN Hot method is usually used for the detection of Mycobacterium

tuberculosis.
For Mycobacterium leprae, ZN Cold method is used. For the cold method
20% Sulfuric Acid is replaced by 5% Sulfuric Acid and Carbol Fuschin is
applied to the smear without heating.

Morphology of different bacteria

• Streptococcus:
▪ Streptococci are spherical or ovoid and are seen in pairs or as chain-like
structures under a microscope.
▪ They are Gram-positive and non-motile, and do not form spores.
▪ Some strains are capsulated

• Staphylococcus:
• Spherical shape (cocci).
• Occurs in clusters resembling grape-like clusters.
• Individual cells may vary in size.
• Gram-positive (in young cultures).
• Non-spore-forming.

• Pseudomonas:
Pseudomonas is a rod-shaped, slender (0.5 to 0.8 μm by 1.5 to 3.0 μm)
Gram-negative organism. It is motile by polar flagella, and sometimes more than two flagella
may be present
• Mycobacteria:
▪ Rod-shaped (0.2-0.6 μm wide and 1.0-10 μm long)
▪ Non-motile (except for Mycobacterium marinum)
▪ Gram-positive
▪ Catalase-positive
▪ Do not form spores
▪ Possess capsules
▪ Most are aerobic, although some are microaerophilic
 • Clostridium tetane: Rod-shaped and up to 2.5 μm long, but they become enlarged
and tennis racket- or drumstick-shaped when forming spores.

• Corynebacterium diphtheriae: Thin, slender gram-positive bacilli, but, especially in

old cultures, they are easily decolorized. They measure about 3-6 μm × 0.6-0.8 μm

Preparation of Culture Media

➢ Nutrient agar:

S.N Ingredients Gram/liter

1. Peptone 5.0

2. Yeast extract 1.5

3. Beef extract 1.5

 Sodium 5.0
4.
chloride

5. Agar 15.0

Final pH at 25°C: 7.4 ±0.2

1. In a beaker, 28 grams of the dehydrated powder or lab-prepared media is added to 1000

milliliters of distilled or deionized water.
2. The suspension is then heated to boiling to dissolve the medium completely.
3. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes.
4. Once the autoclaving process is complete, the beaker is taken out and cooled to a temperature
of about 40-45°C.
5. If enrichment is desired, the addition of blood or biological fluids can be done after the
autoclaving process.
6. The media is then poured into sterile Petri plates under sterile conditions.
7. Once the media solidifies, the plates can be placed in the hot air oven at a lower heat setting
for a few minutes to remove any moisture present on the plates before use.

➢ Nutrient Broth: Basically, the nutrient broth is the nutrient agar that lack of the solidifying
agent, agar powder. They remain in liquid form at room temperature and are usually used to
maintain the stocks of microorganisms. In general, they are used to grow fastidious
organisms. Also, you can enrich your nutrient broth with blood, serum, sugars… etc for
special purposes.

1. Add 13g of nutrient broth powder (CM0001B) in 1L of distilled water.

2. Mix and dissolve them completely.
3. Pour them into the final containers (eg. conical flask)
4. Sterilize by autoclaving at 121°C for 15 minutes.

➢ Blood Agar: Blood agar, like most other nutritional media, has one or more protein sources,
salt, and beef extract for vitamins and minerals. Besides these components, 5% defibrinated
mammalian blood is also added to the medium. The blood agar base is commercially sold by
various vendors, or it can also be prepared in the laboratory if the necessary ingredients are
available.
Components:

S.N Ingredients Gram/liter

1. Peptone 10.0

2. Tryptose 10.0
 Sodium 5.0
3.
chloride

4. Agar 15.0

Final pH at 25°C: 7.3 ±0.2

1. About 40 grams of the prepared medium is added to 1000 ml distilled or deionized water.
2. The suspension is heated up to boiling to dissolve the medium completely.
3. It is then sterilized by autoclaving it at 15 lbs pressure and 121°C for about 15 minutes.
4. The medium is then taken out of the autoclaved and cooled to about 40-45°C.
5. To this, 5% v/v sterile defibrinated blood is added aseptically and mixed well.
6. The media is then poured into sterile Petri plates under sterile conditions.
7. Once the media solidifies, the plates can be placed in the hot air oven at a lower heat setting
for a few minutes to remove any moisture present on the plates before use.

➢ Peptone Water: It is a broth medium used for the growth of the organism and a base for
determining carbohydrate fermentation patterns of non-fastidious organisms. In addition, it is
also used for the detection of indole production by the organism.
Components:

Ingredients Gms / L

Peptone 10.0

Sodium chloride 5.0

Final pH (at 25°C) 7.2±0.2

1. Suspend 15.0 grams in 1000 ml distilled water.

2. Mix thoroughly and distribute into the final containers.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

➢ MacConkey Agar:

Ingredients Amount

Peptone (Pancreatic digest of gelatin) 17 gm

Proteose peptone (meat and casein) 3 gm

Lactose monohydrate 10 gm

Bile salts 1.5 gm

 Ingredients Amount

Sodium chloride 5 gm

Neutral red 0.03 gm

Crystal Violet 0.001 g

Agar 13.5 gm

Add to make 1
Distilled Water Liter

Final pH 7.1 +/- 0.2 at 25 degrees C.

1. Suspend 49.53 grams of dehydrated medium in 1000 ml purified/distilled water.

2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45-50°C.
5. Mix well before pouring into sterile Petri plates.

❖ Basic Concepts for Specimen Collection

• Collect the specimen from the actual site of infection, avoiding contamination from adjacent
issues or secretions.
• Collect the specimen at optimal times (for example, early morning sputum for AFB culture).
• Collect a sufficient quantity of material. Use appropriate collection devices: sterile, leak-proof
specimen containers. Use appropriate transport media (anaerobe transport vials, e-Swabs for
bacterial culture, Cary-Blair for stool culture, VTM for viral and Chlamydia cultures, and
urine boric acid transport for bacterial urine cultures). Check expiration date before
inoculating collection device.
• Whenever possible, collect specimens prior to administration of antimicrobial agents.
• Properly label the specimen and complete the order in Epic. The specific source of specimen
is required. Example: wound, left leg.
• Minimize transport time. Maintain an appropriate environment between collection of
specimens and delivery to the laboratory.
• If appropriate, decontaminate the skin surface. Use 70-95% alcohol (ALC) and 2%
chlorhexidine or 1-2% tincture of iodine (TIO) to prepare the site. Allow a contact time of two
minutes to maximize the antiseptic effect.
• For the orders with more than one test, ensure that the proper transport is utilized. For
example, anaerobic culture requests need to be submitted in anaerobic transport media;
bacteriology requests should not be in viral media; AFB requests should not be in anaerobic
transport media and swabs will not be accepted.
Urine Specimen Collection:
• Clean genital area with the wipes. FEMALES: Clean from front to back; MALES:
Clean urethral opening area.
• Allow first urine flow to go into the commode.
• Catch the "mid-stream" of the urine in the sterile container.
• Replace lid firmly on the container.
• Label specimen with name, date and time of collection.
• Place the specimen in the transport bag.
• Transport the specimen to the lab as soon as possible. If delay is necessary, specimen
may be kept in your refrigerator up to 24 hours.

PUS specimen collection: A sterile swab may be used to collect cells or pus from a superficial
wound site. From deeper wounds, aspirations of fluid into a syringe and/or a tissue biopsy are the
optimal specimens to allow for the recovery of aerobic and anaerobic bacteria.

Method of Inoculation
Inoculation is the process of adding bacteria to a culture media so they can reproduce
there. To increase immunity against a certain disease, it is frequently used to introduce
vaccines, serum, or other antigenic substances into the body. Inoculating loops and needles
are hand-held and compact appliances that introduce microorganisms like bacteria or
yeast into plated or tubed growth media before incubation, multiplication, or growth.

Parts of Inoculating Loops and Needles:

 1. Handle: The inoculating loop or needles are handled by holding the handle part of the loop or
needle and facilitating fatigue-free application. It is made of aluminum and brass in either an
insulated or non-insulated format. The handle is 8 inches long.
2. Shaft: Nickel-plated brass is used to make the loop shaft. For added heat protection for the
user, PVC is used to insulate the shaft of the loop handle.
3. Turret: It holds nickel-chromium or platinum wire.
4. Loop: The nichrome or platinum wire affixed to the turret is twisted into a loop structure at
the end. The wire is resistant to high temperatures and oxidation. The loop is 2mm to 5 mm
in diameter.
5. Needle: It is substantially identical to the loop but uses a straight wire instead.
Inoculating Loops and Needles Operating Procedure:
1. Sterilize the wire inoculating loop by angling through the heat source until the full length of
the wire begins to glow red/orange from the heat.
2. Cool the loop or needle before selecting the bacterial sample to be transferred.
3. The loop/needle is reheated after use to sterilize it.
4. Disinfect a 12-inch diameter ring around the base of the heating device to eliminate any
bacteria that survived the heating process.

Applications of Inoculating Loops and Needles:

1. Inoculating needles are used in microbiology to investigate bacteria and fungi on semi-solid
media.
2. Needle-oriented culture techniques are also used in biotechnology, cell biology, and
immunology.
3. The inoculation loop/ needle is used for streak plate streaking, fishtail inoculating slant
cultures, and inoculating stab cultures.
4. Stab cultures are used to research cell motility, microbial oxygen requirements utilizing
thioglycolate cultures, and the gelatin liquefaction of bacteria. Stab cultures especially call for
the inoculation needle.
5. Inoculating loops and needles are employed for inoculation, serial dilution, sterile sampling,
transfer, and spreading microbiological samples.

Types of Media Used for Inoculation:

• Agar Plates
• Broth Culture
• Slant culture
• Plate culture
• Stab culture

❖ Agar Plates:
Agar plates are some of the most common media which are in use for growing bacteria and
other microorganisms. A mixture of agar and nutrients necessary for bacterial growth. This is
then poured into circular Petri dishes where the agar solution solidifies. After this,
 inoculation of a solution-containing microorganism onto these plates with the help of
streaking.
A small streaking loop is a dip into a solution, which contains bacterial cells that are used to
streak onto the plates with the bacteria. These plates are stored at the proper temperature for
bacterial growth for further study. We can also inoculate liquid media suspensions of bacteria
to grow and reproduce.
A single culture of microorganism added to a small solution to form a mixture and pipette
into liquid media. For Bacterial growth, we need media in which the mixture of
microorganism and solution contain nutrients, compounds, and other necessary molecules.

▪ Streak Plate Method

This method is used to obtain completely isolated colonies from a culture or specimen inoculum
through the creation of sections of increasing dilution on a single plate.

• Inoculate clinical specimens through the use of sterile inoculation loops into the agar media.
Spread the specimen gently on a section of the culture media surface
• Extract loop from the inoculated area and distribute into a second part
• Extract the loop from the other section and disperse it to the 3rd section. Continue for the 3rd
and 4th section. Make sure that sections 1 and 4 are not overlapping. Unload inoculation loop
used into suitable containers
• Substitute the lid followed by incubating the streaked agar plate at the optimum temperature
(inverted stance), so as to curb condensation

▪ Pour Plate Method:

The pour plate method is a laboratory technique for isolating and counting viable
microorganisms like bacteria and fungi in a liquid sample that is added to a molten
agar medium. In general, this technique counts the total number of CFUs (colony-
forming units) on the surface of the solid medium.
 • Serial dilution – If the sample is a liquid, it can be diluted serially with sterile broth
or distilled water. If the sample is semisolid or solid, it must first be emulsified
before being serially diluted to reduce the microbial load to the permissible limits.
• In the pour plate method, the sample is either added to the Petri plate and then
poured with the molten agar medium, or the sample is mixed with the molten agar
medium before pouring.
• Now the medium is allowed to solidify before being incubated at the appropriate
temperature to grow the microbes present in the sample. The number of isolated
colonies is counted after incubation.
The major difference between the streak and pour plate method is that in the streak
plate, the melted agar is added first, followed by a loop of bacteria from a slant,
whereas in the pour plate, the bacterial broth is added first, followed by the agar.

❖ Spread Plate Method:

It is used for evenly spreading cells to ensure growth of the isolated separate
colonies. Further, it can be used for serial dilutions. The spread plate method is used
for enrichment, enumeration and screening and selection of microorganisms.
• Onto the agar media, with the help of a sterile spreader, inoculate the clinical
specimen where we spread the bacteria gently on the whole culture media
surface. This is done by rotating the plate while spreading it backwards and
forward. Refrain from allowing the spreader to touch the edges of the plate
• Substitute the lid and ensure the plate is standing in an upright position for
drying (10-12 minutes)
• Now incubate the spread agar plate at the optimum temperature with the lid at
the base (inverted)
The biggest advantage of a spread plate method is that the morphology of the
isolated bacteria can be seen vividly. The only disadvantage is that sometimes fungal
colonies may grow.
Broth Culture:
An inoculation needle used in inoculating a sterile broth culture. Flaming the open end of the
broth will keep it sterile. The broth moved up to the needle so that the tip of the needle is
submerged while maintaining the original position of the needle.
Swirling of the needle carefully can help the inoculation of the microorganism from the
needle to the sterile broth. The inoculated broth culture then removed from the needle. With
the help of the Aseptic technique which is applied to the open end of the broth culture so as
to prevent contaminants, and the needle is flamed for sterilization.

Slant Culture:
To inoculate a slant culture in a fishtail inoculation technique we use inoculation
needle. After transferring the microorganisms from the original microbial culture to the
inoculation needle the sterile slant culture is uncapped. The open end of the uncapped slant
culture is then flamed.
The position of the slant is such that it moves the needle up until the tip of the inoculation
needle comes in contact with the base surface of the sterile media. A zigzag pattern is formed
on the agar surface when the inoculation needle inoculates the sterilize agar by the
manipulation of the media. To remove the inoculation needle, we use an aseptic technique.

Stab Culture:
In inoculating a stab culture, an inoculation needle is an essential tool. Removal of a sterile
stab culture cap is completed and the open end of the needle is flamed. The needle tip and
length of the needle is pushed into the stab media until the needle reaches 0.5 inches away
from the bottom of the stab media. The inoculation needle is removed from the media in the
same direction and path that it was pushed into the stab media to prevent the wobbling effect
that may disturb the culture. We can sterilize the needle with the help of a flame.

❖ Agar stab technique:

It is used in the preparation of stab cultures, from a plate select single colonies.
• Select a well-isolated colony through aseptic technique using an inoculating stab
needle (sterile) and stab it a few times via the agar to the base of the tube
• Substitute the cap and secure loosely during incubation enabling exchange of gases
• Incubation of this stabbed plate at the suitable temperature is carried out