Microbiology

Microbiology

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Microbiology

By

Dr. Saurabh Arora

Founder : Lab-Training.com
Director : Auriga Research Ltd., Arbro Pharmaceuticals Ltd. (Analytical Division)
E-mail : saurabharora@arbropharma.com

&

Dr. Rajshree Saxena

Auriga Research Ltd.

And
Arbro Pharmaceuticals Ltd.
Analytical Division,

4/9 Kirti Nagar Industrial Area, New Delhi - 110015 (INDIA)

Microbiology

Authors Profile
Dr. Saurabh Arora
Holds a PhD in Pharmaceutics and has invented a patented nano technology based delivery system for curcumin the active
constituent of

turmeric or haldi in local language. .

Has a number of national and international research publications and patents to his credit.
Heading the testing laboratory and research business at Arbro and Auriga for close to 10 years.
Has designed and setup 4 state of the art testing laboratories in New Delhi, Baddi and Bangalore.
Leading a team of over 250 scientists and professionals in 4 laboratories which serve more than 10,000 customers each year
from the food, retail, hospitality, nutraceutical, pharmaceutical, cosmetics, agri, medical device, research, academics and
real-estate industries.
Firm believer that sharing knowledge and experience is the only way to move the society further.
Established www.lab-training.com in June 2011 to share his teams expertise in the field of testing with scientists and
students around the world, the site today has over 10,000 subscribers and offers online training courses on various
techniques used in testing laboratories across industries.
He also established www.foodsafetyhelpline.com in January 2013 as a one stop solution for the people in the food industry
to stay up-to-date, understand and implement the requirements of the Food Safety and Standards Act and the Food Safety
and Standards Authority of India (FSSAI). The site has a simple objective to help food businesses understand and comply
with the requirements of this new and rapidly evolving food law which has been put in place to provide safe and hygienic
food to all the citizens of India.
He also regularly speaks and presents at industry meets, conferences, workshops and webinars on a variety of topics related
to the testing and quality of products.
He has completed a training on Lean Labs: Lab Performance Optimisation and has been implementing the principles of
Lean Six Sigma for labs which has helped the business grow at over 30% each year.

Microbiology

Awards
He received the Award for 10 best training and development practices in private sector on behalf of Arbro
Pharmaceuticals Ltd. At the Excellence in Training and Development Awards 2013.
Arbro was also adjudged among the top 100 SMEs of India at the BOI India SME 100 Awards in 2012
Dr Rajshree Saxena has earned her Doctorate degree in Biotechnology from Amity Institute of Biotechnology, Amity
University, Noida, U.P. She has a Masters in Microbiology from Rani Durgavati Vishwavidyalaya, Jabalpur (M.P). She has
about 9 years of pre and postdoctoral experience, both in industry as well as in research and teaching. Her field of expertise
is microorganisms and their exploitation for waste management, various industrial and medical applications.
Dr Rajshree Saxena has authored 1 book in Microbiology, co-authored 13 papers that have been published in peer reviewed
journals, 1 book chapter and about 9 papers that have been presented in various national and international conferences.

Currently she works as a scientific content writer.

Microbiology

Lab-Training.com

Knowledge grows when shared with others.

Knowledge does not discriminate between national boundaries, color of skin, religion, caste, gender and creed.
Our firm belief in these principles has contributed immensely to the growth of our web based portal through
sharing of our expertise and skills

Our world class infrastructure, manpower skills and over 25 years of experience is now accessible through web based portal
as we moved on from limited classroom training provider role over the last few years.
Our e-learning courses, articles and certificate programmes have been appreciated by industries, institutions, regulatory
organizations and even individuals across the globe. There are constant demands for courses and articles on techniques of
analytical interest and improvement of laboratory activities. We are bound to upgrade our content keeping the needs of our
clients and followers in mind. It will be our endeavor to provide leadership in this key area of development.

Microbiology

Contents
Chapter 1 : Introduction to Microbiology
Chapter 2 : Observing Microorganisms- Microscopic Examination
Chapter 3 : Observing Microorganisms-Staining
Chapter 4 : General Classification and Characterization of Microorganisms
Chapter 5 : Prokaryotic Cell-Brief Description of Structure and Function
Chapter 6: Conditions Favourable to Growth of Microorganisms
Chapter7:Cultures- the Growth and Survival Media of Microorganisms
Chapter 8 : Activities and Practices in a Microbiology Laboratory
Chapter 9 : Beneficial and Detrimental Roles of Microorganisms
Chapter10 : Microbiology in Food Industry
Chapter 11 : Microbiology in Pharmaceutical Industry
Chapter 12 : Common Interview Questions for Microbiologist with answers
Chapter 13 : Conclusions

Microbiology

Introduction to Microbiology
Microbiology is the study of microscopic living organisms, the microbes that are usually too small to be seen with the
unaided eye. Microorganisms are extremely diverse and ubiquitous in nature. Microbiology deals with the study of
distribution of microorganisms in nature, their unique metabolic characteristics, their relationship with each other and with
other organisms, their effect on human beings, other animals and plants,etc.

Microbes include bacteria, fungi (yeasts and molds), protozoa, microscopic algae, and also viruses (the noncellular entities
regarded as straddling the border between life and nonlife).

Bacteria are unicellular organisms whose genetic material is not enclosed in a special nuclear membrane (hence called
prokaryotes). Bacterial cells generally appear in shapes as Bacillus (rodlike), coccus (spherical or ovoid), and spiral
(corkscrew or curved) are among the most common shapes, but some bacteria are starshaped or square. They may
appear in pairs, chains, clusters, or other groupings.

Fungi are eukaryotic organisms with cells that have a defined nucleus and rigid cell walls. Fungi may be unicellular
(yeasts) or multicellular. The most typical fungi are molds that consists of mycelia, which are made up of long
filaments called hyphae that branch and intertwine. Large multicellular fungi include mushrooms that may resemble
plants, but they cannot carry out photosynthesis and are saprophytic or parasitic in nature.

Protozoa are eukaryotic unicellular organisms that move by cilia, flagella or pseudopodia, and are usually parasitic
(organisms that derive nutrients from living hosts) or free living in nature. Their size varies from 5-200 m, they lack a

Microbiology

rigid cell wall and are differentiated on the basis of morphological, nutritional and physiological characteristics.
Protozoa are known to cause various diseases in human beings and animals.

Algae are simple photosynthetic eukaryotes found most commonly in aquatic environments or in damp soil. They are
unicellular to multicellular and either motile or nonmotile. Extensive algal growth in water bodies deteriorate the
quality of the water releasing toxic chemicals into water bodies, or growing in swimming pools.

Viruses are a distinctive group of acellular obligate intracellular parasites, very small in size, whose uniqueness resides
in their simple, acellular organization with inimitable pattern of reproduction. A complete virus particle consists of one
or more molecules of DNA or RNA enclosed in a coat of protein, and sometimes also in other complex layers that
contain carbohydrates, lipids, and additional proteins.

Microorganisms thrive in almost all types of environmental extremities in nature. Microorganisms are closely associated
with our day to day lives in beneficial as well as detrimental ways. Fortunately most microorganisms are completely
harmless to humans and only a very small fraction of the community belongs to the pathogenic group. Moreover, the
bacteria are highly specific in nature as far as their pathogenecity towards a host is concerned.
Many microorganisms are known to cause mild or severe infectious and fatal diseases in humans and animals.
Microorganisms also cause food spoilage, deterioration of materials such as wood, metal etc. However, they play a major
role in making of various food products and beverages as yogurt, cheese, wine etc. Microorganisms also contribute towards
the production of various antibiotics, and biomolecules as enzymes, vitamins that are used in medicinal and therapeutic
purposes.
Points to Remember:
1.

Microbiology may be defined in terms of the size of the organisms studied and the techniques employed.

2.

Antony van Leeuwenhoek was the first person to describe microorganisms.

3.

If an object has a diameter 0.1 mm or less, eye cannot see it and very little details can be seen in an object having
diameter 1 mm. So roughly speaking organisms having diameter 1 mm or less are called microorganisms and are
studied in Microbiology.

4.

Size range of molds is 2.0-10 m and yeast has size varying in the range of 5-10 m.

5.

The cottony growths sometimes found on bread and fruit are mold mycelia.

6.

Prokaryotic cells differ from eukaryotic cells in lacking a membrane-delimited nucleus, and in other ways as well.

Microbiology

Observing Microorganisms-Microscopic
Examination
Microbiology deals with the study of microorganisms that cannot be seen distinctly with the unaided eye. Considering the
nature of the objects to be studied, the microscope becomes an instrument of paramount importance. Modern microscopes
produce images with great clarity, magnifications that range from ten to thousands of times.
Compound Light Microscopy
This is the most basic microscope used for studying microorganisms. It consists of a series of lenses that utilizes visible
light as its source of illumination. Various small specimens can be studied to fine details with a compound light microscope.

Labeled Diagram of a Compound Microscope

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Microbiology

Components of a Compound Microscope

The major components of a compound microscope are :
Framework: The basic frame structure is made up of metal, which includes the arm and base to which whole of the
magnification and optical components are attached. The metallic arm is connected to a U shaped strong and heavy base that
provides stability to the instrument.
Stage: this is the flat horizontal platform positioned at about halfway through the length of the microscope with a hole at the
centre that allows the passage of light for illumination of the sample.
Focus knobs: Two pairs of knobs are attached to the arm that help in up and down movement of the stage and in adjustment
and focusing of specimens of different thickness.
Lens Systems: All microscopes employ a set of different types of lens systems: the oculars, the objectives, and the
condenser, that have different focusing power, and contribute to the complete magnification system.
Nose piece: A revolving nosepiece which holds the objectives is attached to the curved upper part of the arm of the
microscope. The nosepiece can be rotated to position the objective with the required magnification in path of the
magnification system, beneath the body assembly and the eye piece.
Eyepiece (ocular lens): The eyepiece or ocular lens is a set of lenses held in a cylindrical tube kept inserted in a tubular
structure on the curved upper part of the arm, above the nose piece. It consists of two or more lenses which focus the image
into the eye. The newest microscopes consist of a pair of eye pieces that allows the observer to use both the eyes to observe
the specimen in the microscope. Such microscopes are called binocular microscopes. The normally used eye pieces have 2X,
50X and 10X magnifications.
Objective: The objectives are usually small cylindrical objects containing a single or a set of lenses attached to the
nosepiece. The nosepiece holds three to five objectives, which contain lenses of varying magnifying power (2X-400 X). The
total arrangement of the lenses is parfocal, which means that the sample stays in focus even when the lenses are changed
from one to another in a microscope.
Condenser: A condenser is also a lens which is fixed below the stage and it focuses the beam of light coming from the light
source onto the slide. The condenser is usually aided with diaphragm and/or filters, to control and manage the quality and
intensity of the light passing through the sample.

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Microbiology

Light Source: The light source is mounted at the base of the microscope. The source of light may be the day light, a
halogen light, or even LEDs and lasers, as used in the latest microscopes. The microscopes have some provision for
reducing light intensity with a neutral density filter.
Types of Compound Microscopes

1.

The Bright-Field Microscope

2.

Dark Field Microscope

3.

Phase-Contrast Microscope

4.

The Differential Interference Contrast Microscope

5.

The Fluorescence Microscope

6.

Confocal Microscope

7.

Two-Photon Microscope

All these types of microscopes yield a distinctive image and may be used to observe different aspects of microbial
morphology.
Points to Remember:

Microorganisms are too minute in size to be seen by unaided eyes, hence are observed and studied using microscopes.

Compound microscopes are commonly used in research labs and institutes to study microorganisms. They use glass
lenses to bend and focuses light rays and produce enlarged images of small objects.

Microscopes are delicate and very expensive instruments in any academic or research institutes. Even the most basic
compound microsopes require a lot of investment. They, hence, need critical care in usage and handling.

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Microbiology

Observing Microorganisms-Staining

Microorganisms are observed and studied with the help of microscopes. However the bacteria have nearly the same
refractive index as water, which makes them visually opaque. Hence, the microbial cells are routinely stained for clear
microscopic examination. Staining is thus an auxiliary technique used in microscopy and microbiology to enhance contrast
in the microscopic images of the microorganisms. Different types of stains and staining procedures are available today to
study the multiple properties of various microorganisms.
Stains used in different staining procedures are aqueous or alcoholic solution of chemical substances known as dyes, which
may be natural or synthetic. These stains adhere to the cell and give it colour and contrasts, making the cell more visible.
The stains may be acidic, basic or neutral. The acidic dyes as picric acid, acid fuschin, eosin etc., stain the cytoplasmic
components of the cells which are basic in nature, while the basic dyes as methylene blue, crystal violet, safranin etc., stain
the acidic components of the cell as nucleic acids. As the bacterial cell is slightly negatively charged on its surface at neutral
pH (pH 7), the basic dyes stain bacterial cells as a whole.
The basic staining process involves fixing or mounting of the sample on to the slide, immersing the sample in dye solution
for a specified time period and then finally rinsing and observing the sample under the microscope. In some staining
procedures, an additional chemical called mordent is added to the stain which increases the interaction between the cell and
the dye forming an insoluble coloured precipitate, which is retained even after the dye is washed away from the cell.
There are basically two types of staining procedures, simple and differential.

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Microbiology

Simple staining is the most basic staining performed to determine cell shape, size, and arrangement of bacterial
cells. This staining technique stains the bacterial cell. It can be performed with basic dyes with different exposure times.
Negative staining is also a simple staining technique, that provides the simplest and probably the quickest technique to get
information about the cell shape, cell breakage, and refractile inclusions in cells such as sulphur and poly hydroxyl
butyrate granules and endospores. The bacteria rendered colourless against a coloured background in negative staining. The
dyes used in the negative staining are usually anionic in nature and hence repelled by negatively charged cytoplasm, leaving
the cells unstained.
Differential Staining is a broad term that is used to describe staining processes which use more than one chemical stain.
Differential staining entails use of a series of dyes to stain the different cell organelles based on the structural and
compositional differences. This type of staining helps identification and categorization of the cells into different groups.
Various differential staining techinques used in microbial studies are
1.

Gram staining

2.

Acid fast staining

3.

Endospore staining

4.

Capsule staining

5.

Flagella staining

6.

Cellwall staining

7.

Nuclear material staining

Points to Remember:

A stain is a chemical that adheres to structures of the microorganism and in effect dyes the microorganism so the
microorganism can be easily seen under a microscope.

Basic stains as methylene blue, crystal violet, safranin and malachite green. are cationic, have positive charge and stain
chromosomes and the cell membranes of many bacteria.

Acid stains as eosin and picric acid are anionic, have a negative charge and stain cytoplasmic material and organelles
or inclusions.

A differential stain consists of two or more dyes and is used in the procedure to identify bacteria.

Most commonly used differential stains for bacterial identification are the Gram stain, the Ziehl-Nielsen acid-fast stain
and endospore stain

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Microbiology

General Classification and Characterization of

Microorganisms
Classification of living organisms is referred to as taxonomy and it aims to classify living organisms by differentiating them
and establishing relationships between groups of organisms.
The kingdom of living organisms was termed as domain by Carl R. Woese in 1978.Organisms are grouped in three domain
systems namely eukarya, archaea and bacteria, primarily on the basis of cell type. Other than the cell type, differences in
rRNA, the three domains differ in membrane lipid structure, transfer RNA molecules, and sensitivity to antibiotics also
account for classification of organisms.

The Domain bacteria includes all of the pathogenic and non-pathogenic prokaryotesThey have a cell wall
composed of peptidoglycan and muramic acid. They also have membrane lipids with ester-linked,
straight-chained fatty acids that resemble eukaryotic membrane lipids. Bacteria also have plasmids, which are
small, double-stranded DNAmolecules that are extrachromosomal.

Domain archaea include three major groups namely methanogens, extreme halophiles and hyperthermophiles.
They lack muramic acid in the cell walls.

Eukarya includes the eukaryotic organismsas animals, plants, fungi, and protistsand have a defined nucleus and
membrane bound organelles

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Microbiology

The taxonomic classification scheme for prokaryotes is elaborated in Bergeys Manual of Systematic Bacteriology which
describes the classification of prokaryotes into two domains: Bacteria and Archaea.
Further classification of domain is as follows

Domain

Kingdom

Phyla

Class

Order

Family

Genus

Species

The binomial (scientific) nomenclature assigns each microbe 2 names Genus noun, always capitalized and species
adjective, lowercase. Both are written italicized or underlined. Ex. Staphylococcus aureus (S. aureus), Escherichia coli (E.
coli)
The basic taxonomic group in microbial taxonomy is the species. Taxonomists working with higher organisms define their
species differently than microbiolo- gists. Prokaryotic species are characterized by differences in their phenotype
andgenotype. Phenotype is the collection of visible characteristics and the behavior of a microorganism. Genotype is the
genetic make up of a microorganism

Micobial Taxonomy

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Microbiology

Bergeys Manual provides classification scheme for bacteria as well as a reference for identification of different bacteria in
the laboratory. According to Bergeys manual of determinative bacteriology, the bacteria are identified on the basis of
morphological characteristics staining, biochemical tests, serology, G-C content, DNA probesand different molecular tests.
In addition to the properties listed in Bergeys, the source and habitat of microorganisms is also important to consider when
identifying microorganisms.
Points to remember:
1.

Taxonomy is the science that deals with classificationof living organisms on the basis of characteristics that are similar
to and different from other organismsand establishing relationships between groups of organisms.

2.

All living organisms are grouped in three domain systems namely eukarya, archaea and bacteria, primarily on the basis
of cell type.

3.

The taxonomic classification scheme for prokaryotes is elaborated in Bergeys Manual of Systematic Bacteriology
according to which the prokaryotes are divided into two domains: Bacteria and Archaea

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Microbiology

Prokaryotic Cell-Brief description of Structure and

function
Prokaryotic cells fall into a size range of about 15m and hence can be observed clearly by microscopes. However, some
prokaryotic cells may be larger than this.
A prokaryotic cell contains external and internal structures. Capsule, flagella, axial filaments, fimbriae, and pili are present
external to the cellwall, while interior of the bacterial cell contains cytoplasm.

Flagella - Flagella are whip like structures made of protein and provide motility to the cell. Prokaryotic cells may be

Monotrichous Cells that have one flagellum.

Lophotrichus Cells that have a clump of flagella known a tuft, at one end of the cell.

Amphitrichous Cells that have flagella at two ends of the cell.

Peritrichous Cells that have flagella covering the entire cell on the surface.

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Microbiology

Fimbriae and pili Fimbriae are proteinaceous, sticky, projected structure used by cells to attach to each other and to
objects around them, while pili are tubules that are used to transfer DNAfrom one cell to another cell.
Capsule Depending on the type of bacterium, there may be an exterior surrounding layer.such as a capsule or slime layer,
made of glyocalyx
Cellwall The prokaryotic cells cell wall is present outside the plasma membrane. It provides rigidity to the cell shape and
structure and protects the cell from its environment. Bacterial cell wall is primarily composed of peptidoglycan and on the
basis of cell wall composition the bacteria classified into gram-positive and gram negative organisms.
Cytoplasmic Membrane The cytoplasmic membrane is a membrane that provides a selective barrier between the
environment and the cells internal structures.
Cytoplasm
Cytoplasm is thick.aqueous, semitransparent, and elasticsmifluid present inside the prokaryotic cell. It is about 80% water
and contains primarily proteins (enzymes), carbohydrates, lipids, inorganic ions, and many low- molecular-weight
compounds. Inorganic ions are present in much higher concentrations in cytoplasm than in most media.
Nucleoid/Genetic material - The cytoplasm also contains a region called the nucleoid, which is where the DNA of the cell
is located. The prokaryotic cell consists of a chromosome that isnt contained within a nuclear membrane or envelope. The
nucleoid or bacterial chromosome comprises a closed circle of double stranded DNA, many times the length of the cell and
is highly folded and compacted.
Ribosomes - Ribosomes are the principle structure in a prokaryotic cell after the nucleoid. They are composed of a complex
of protein and RNA, and are the site of protein synthesis in the cell. The prokaryotic ribosomes are 70S, comprised of sub
units 50S and 30S (S stands for the sydberg coefficient which is a function of their size and shape, and determined by their
rate of sedimentation in a centrifuge)
Inclusion bodies - Many granular structures known as inclusion bodies are found in the cytoplasm of certain bacteria.
These contain organic compounds such as starch, glycogen or lipid and act as food reserves. Some sulphur and
polyphosphate containing bodies are also found and are known as volutin or metachromatic granules.
Endospore - A number of gram-positive bacteria can form a special resistant, dormant structure called an endospore.
Endospores develop within vegetative bacterial cells and are extraordinarily resistant to environmental stresses such as
heat,ultraviolet radiation,gamma radiation, chemical disinfectants, and desiccation.

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Prokaryotic cells are much smaller than eukaryotic cells. The main differences between prokaryotic and eukaryotic cells are
described below:

Characteristics

Prokaryotic Cells

Eukaryotic Cells

Cell wall

Complex composition in layers,

typically contains peptidoglycan

Composition is simple,
peptidoglycan not found

Plasma membrane

No carbohydrates or sterols

Contains carbohydrates and sterols

Glycocalyx

Present as capsule or slime layer

Present in cells that lack cellwall

Cell organelles

None. Only some inclusion bodies are ER, golgi body, lysosomes,
present
mitochondria, lysosomes

Nucleus

Not well defined, without any nuclear Well defined nucleus present, with
membrane or nucleoli
nuclear membrane and nucleus

Chromosome

Single circular chromosome present as Multiple linear chromosomes found

nuclear material without histones
with histones

Ribosomes

70S

80S

Cell division

Binary fission

Mitosis

Points to Remember:
1.

All bacteria are prokaryotes and much simpler structurally than eukaryotes.

2.

Most bacteria have a cell wall outside the plasma membrane to give them shape and protect them from osmotic lysis.

3.

Bacterial walls are chemically complex and usually contain peptidoglycan or murein. Bacteria often are classified as
either gram positive or gram negative based on differences in cell wall structure and their response to Gram staining

4.

The cytoplasmic matrix contains inclusion bodies and ribosomes.

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Microbiology

5.

Prokaryotic genetic material is located in an area called the nucleoid and is not enclosed by a membrane.

6.

Structures such as capsules, flagella, and sex pili are found outside the cell wall.

7.

Some bacteria survive adverse environmental conditions by forming endospores, dormant structures resistant to heat,
desiccation, and many chemicals.

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Microbiology

Conditions Favourable to Growth of

Microorganisms

Microorganisms

All living organisms, microorganisms require a combination of various physical and chemical factors for their growth and
multiplication. Although individual cells approximately double in size during their lifetime this change is not very
significant. Microbial growth actually refers to increase in numbers of the cells. The requirements for microbial growth can
be divided into two main categories: physical and chemical.

Physical aspects include temperature, pH, and osmotic pressure.

Chemical requirements macromolecules (carbon, nitrogen, hydrogen, sulfur, phosphorus, oxygen) and micro
molecules (trace elements and organic growth factors as magnesium, potassium, sodium, calcium and iron in their
ionised forms)

Physical Requirements
Temperature: Microorganisms are classified into three primary groups on the basis of their preferred range of temperature:

psychrophiles (cold- loving microbes),

mesophiles (moderate-temperature-loving microbes),

thermophiles (heat-loving microbes).

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pH: pH 6.5-7 (neutral range) is best suited for bacterial growth. However some bacteria can grow at an acidic pH below
about pH 4, are called acidophiles. While some bacteria prefer an alkaline pH (8-9), are called alkalophiles. Molds and
yeasts (fungus) require an optimum pH of about 5 to 6 for growth.
Osmotic Pressure: Microorganisms require water for growth as they obtain almost all their nutrients in solution from the
surrounding water. Some organisms adapt to the extreme high salt concentrations are called extreme halophiles, while some
microorganisms require an optimum salt concentration for their growth, are called as obligate halophiles. Some
microorganisms do not require salt for growth but are able to grow in salt concentrations upto 2%, (a concentration that
inhibits the growth of many other organisms). These are called facultative halophiles.
Chemical Requirements

Carbon forms the skeleton or backbone of all organic molecules and hence, is the central component of the biological
macromolecules as it. Hydrogen is also an important molecule that participates in energy generation processes in most
microorganisms. Oxygen is of central importance to the respiration of many microorganisms while nitrogen is needed
for the synthesis of proteins and nucleic acids, as well as for important molecules such as ATP.

In addition to the need for carbon, hydrogen and oxygen, microorganisms also require sources of energy and electrons for
growth to take place. They can be grouped into classes on the basis of their nutritional requirements.
1.

Autotrophs and Heterotrophs: Microorganisms can be classified as either heterotrophs or autotrophs with respect to
their preferred source of carbon. Autotrophs can use

as their sole or principal source of carbon. Organisms that

use reduced, preformed organic molecules as carbon sources are heterotrophs

2.

Phototrophs and Chemotrophs: Phototrophs use light as their energy source, while chemotrophs obtain energy from
the oxidation of chemical compounds (either or- ganic or inorganic). Microorganisms also have only two sources
forelectrons.

3.

Lithotrophs and Organotrophs : Lithotrophs (i.e., rock-eaters) use reduced inorganic substances as their electron
source, whereas organotrophs extract electrons from organic compounds.

All microorganisms come into one of four nutritional classes based on their primary sources of carbon, energy, and
electrons, as demonstrated in the following table

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Microbiology

Major Nutritional types

Source of Energy,
Hydrogen/Electron, Carbon

Examples

Photolithoautotrophy

Light Energy

Algae

Inorganic hydrogen/electron
donor

Purple and Green sulphur

bacteria

Carbon source-

Cyanobacteria

Light Energy

Purple nonsulphur bacteria

Organic hydrogen/electron
donor

Green nonsulphur

Photoorganoheterotrophy

Organic carbon source

Chemolithoautotrophy

Chemoorganoheterotrophy

Chemical (organic) energy

source

Sulphur oxidising bacteria

Inorganic hydrogen/electron
donor

Hydrogen bacteria

Carbon source-

Nitrifying, Iron oxidising

bacteria

Chemical (organic) energy

source

Protozoa

Organic hydrogen/electron
donor

Fungi

Organic carbon source

Non photosynthetic
bacteria

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Points to Remember:

Microorganisms require optimum physical conditions and a combination of various chemical factors for their growth
and multiplication.

Microorganisms can be classified and grouped into 4 major categories on the basis of source of energy they utilize,
hydrogen/electron donor used and source of carbon.

Autotrophs use

Phototrophs use light energy, and chemotrophs obtain energy from the oxidation of chemical compounds.

Electrons are extracted from reduced inorganic substances by lithotrophs and from organic compounds by

as their primary or sole carbon source; heterotrophs employ organic molecules.

organotrophs

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Microbiology

Cultures-the Growth and Survival Media of

Microorganisms
The articial culture and maintenance of microorganisms is the basic necessity of the modern day research. Culturing of
microorganism requires a supply of the necessary nutrients in the form of a culture media, together with the provision of
appropriate conditions such as temperature, pH and oxygenconcentration.A culture medium is a solid or liquid preparation
that contains nutrients that are essential for the growth, transport and storage of microorganisms.
Culture media may be categorised into
1.

Chemically defined media: This is the medium whose exact chemical composition is known. Such media are used in
the research laboratories for growth of specific bacteria. For ex. Glucose is used in media used for the growth of
chemo-heterotrophic E.coli

2.

Complex Media: This is the medium where the exact chemical composition is not known as it is made up of nutrients
including extracts from yeasts, meat, or plants, or digests of proteins from these and other sources. Ex. Nutrient agar- it
is one of the most common complex media used for the growth of heterotrophic bacteria.

3.

Selective and Differential Media: In clinical and public health microbiology, it is very necessary to detect the
presence of specific microorganisms associated with disease or poor sanitation. For this task, selective and differential
media are used.

Selective media favour the growth of particular microorganisms. Selective media are designed to suppress the growth of
unwanted bacteria and encourage the growth of the desired microbes.For example, bismuth sulphite agar (BSA) is one
medium used to isolate the typhoid bacterium, the gram-negative Salmonellatyphi, from faeces. Bismuth sulphite inhibits
growth of grampositive bacteria and other gram-negative intestinal bacteria.
Differential media make it easier to distinguish colonies of the desired organism from other colonies growing on the same
plate. For ex. Blood agar (which contains red blood cells) is used to identify bacterial species as Streptococcuspyogenesthat
destroy red blood cells.
4. Enrichment Culture media: This media is used to enhance the growth of a specific microorganism present in a sample.
It is particularly employed in pathological microbiology. Enrichment medium is usually combined with incubation
conditions (temperature, aerobic/anaerobic, etc) to select for growth of the desired organism. Foe ex. Use of bile salts in
media designed for enrichment of Enterobacteriaceae such as E. coli.

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5. Media for growth of anaerobic bacteria: Anaerobic bacteria require absence of oxygen to grow, hence they must be
placed in a special medium called a reducing medium.These media contain ingredients, such as sodium thioglycolate, that
chemicallycombine with dissolved oxygen and deplete the oxygen in the culture medium.
Pure Cultures
Microorganisms occur as mixed populations in natural habitats. However, pure cultures are needed to characterize and study
any organism thoroughly. Some of the ways to prepare pure cultures are
1. Spread plate method:
In this method, a small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to the center of
an agar plate and spread evenly over the surface with a sterile bent-glass rod.

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2. Streak plate method : In this method a microbial inoculum is transferred to the edge of an agar plate with an inoculating
loop or swab and then streaked out over the surface in one of several patterns.

In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.

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3. Pour plate method : In the pour plate the original sample is diluted several times to reduce the microbial population
sufficiently to obtain separate colonies after plating.

Microbial Growth:
There are four basic phases of microbial growth: the lag phase, the log phase, the stationary phase, and the death phase.

In the lag phase represents the initial state when there is very little or no multiplication of cells. This phase can last
from zero to one hour to several days.

Log phase starts with the logarithmic multiplication of the cells, when the cell number starts increasing exponentially.
At this stage the cells are the most active metabolically.

The stationary phase is the state of equilibrium where the growth rate slows down and the number of dead
microorganisms equals the number of new microorganisms, and the population stabilizes.

In this phase the number of dead cells exceeds the number of new cells. This phase continues until the population is
diminished or dies out entirely.

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The figure below represents a typical growth curve.

Points to remember

Different types of media are used for culturing of different microorganism.

Culture media can be prepared from chemically defined components (defined media or synthetic media) or may
contain constituents like peptones and yeast extract whose precise composition is unknown (complex media).

Culture media are classified based on function and composition as general purpose media, enriched media, selective
media, and differential media.

Pure cultures usually are obtained by isolating individual cells with any of three plating techniques: the spread-plate,
streak-plate, and pour-plate methods

Microbial growth can be represented by a typical curve known as the growth curve.

The growth curve usually has four phases: the lag, exponential or log, stationary, and death phases

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Activities and Practices in a Microbiology

laboratory

Laboratory autoclave for sterilzation

Microbiological laboratories are involved primarily in culturing, examination and identification of microorganisms. These
processes involve a number of techniques that a microbiologist should be well acquainted with.
The basic techniques practiced in a microbiological laboratory are
1. Aseptic techniques and Sterilization:
Autoclave is the instrument used in the microbiology labs for sterilization of all types of media and solutions. At 121C and
pressure 15 lb/sq. in. for 15 minutes, all living organisms including spores are killed in an autoclave.
All the apparatus and glassware are sterilized in a hot air oven at 160 C for 2 hrs or exposure to radiation.
Aseptic techniques are employed to minimize the chances of bacterial contamination. This is accomplished by
disinfection of working areas with disinfectants such as phenols, alcohol, surfactants, detergents, halogens etc., minimising
possible access by bacteria from the air to exposed media, and use of flames to kill bacteria while working on cultures and
inoculation.

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2. Preparation of Microbiological media

Microorganisms have specific nutritional requirement as far as growth is concerned. The two basic media used in
bacteriology are

Nutrient broth, which is a liquid medium used to culture bacteria usually in tubes or conical flasks.

Nutrient agar which is set into a jelly by the addition of a seaweed extract called agar, and the melt on heating is
poured into glass or plastic petri dishes or plates.

Different types of media are used for specific microorganisms.

3.Inoculation and Incubation:
To prepare a culture, the bacteria may be introduced to the media (inoculated) by various means.

A heat sterilized inoculating loop is usually used to introduce a small amount of any microbial culture in a liquid
medium (broth)

On a solid media the inoculation is done using a heat sterilized inoculating loop or a micropipette using sterilized tips.

The techniques include pour plate method, spread plate method and the streak plate method.

The liquid broth or the solid agar media (in petri plates) are incubated i.e. placed in incubators that have a regulated
temperature (usually 37C for bacteria and 25-28C for yeast and molds). The petri plates with bacterial inoculation are kept
in inverted positions to prevent condensation droplets from falling onto the surface of the agar. Petri dishes are kept sealed
with paraffin strips to prevent any contamination and maintenance of the cultures for a longer time.
Petri dishes can be stacked and conveniently stored in the refrigerator
4.Enumeration of Bacteria
Enumeration of microorganisms in a given sample is an important aspect of microbiology. There are different methods
employed for the enumeration of bacteria in a given sample.

Standard (Viable) Plate Count: in this method the samples are serially diluted, plated on an agar plate and incubated for
growth of the colonies. Total number of colonies that grow are counted and multiplied with the dilution factor for
obtaining the number of bacteria present in the sample.

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Direct Microscopic Count: In this method a ruled slide consisting of a counting chamber is used to directly count the
number of microbes present in a sample. Example Petroff- Hausser counting chamber.

Turbidimetric measurement: As the bacteria start growing in a liquid medium after inoculation, the media becomes
turbid and dense. This can be measured using a spectrophotometer which allows/ blocks the passage of light of a given
wavelength.

5. Identification of Microorganisms
Accurate identification of microorganism is very essential in wide ranged applications of microbiology. Identification of
microorganism is done through various methods

Study of morphological characteristics are useful in basic categorization of any microorganism.

Staining is a very important method in identification if microorganisms. Gram staining and acid fast stains are the basic
staining methods used in any microbiology laboratory.

Microscopes are used to observe microorganisms after all types of staining.Living bacteria can also be detected by
direct observation using a light microscope.

Biochemical tests are widely employed for the identification of bacteria. These tests exploit the metabolic properties of
the microorganisms for their identification.

Serological tests as slide agglutination, ELISA, and Western blotting,that involve the reactions of microorganisms with
specific antibodies, are useful in determining the identity of strains and species.

The molecular methods for bacterial identification include % G+C comparisons, PCR, rRNA sequencing, DNA
fingerprinting by restriction fragment length polymorphism, or RFLP, nucleic acid hybridization.

Points to remember:

In a microbiology lab, autoclave is used for sterilization of all media or solutions. Glass wares are sterilized in hot air
ovens

Nutrient broth and nutrient agar are the most common media used for bacterial growth.

Pour plate, spread plate, streak plate methods are employed for obtaining pure cultures

The growth temperature for bacteria is 37C and 25-28C for yeast and molds.

Number of bacteria in a given sample can be obtained by plate count, direct microscopic count or spectrophotometric
method.

Staining is the most primary step in the identification of bacteria

Biochemical tests serve as a major platform for bacterial identification. Serological and molecular methods
are used for confirming the microorganism identification to the generic and species level.

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Beneficial and Detrimental Roles of Microorganisms

Friendly and Unfiendly microorganisms

Microorganisms occupy every small niche of the ecosystem and influence us in many ways. Their influence on human life
is generally beneficial but at times can also be detrimental.
The diversified role of microbes begins at their natural habitats. Elements as carbon, nitrogen, oxygen, sulphur, and
phosphorus are basic requirement for life forms to survive. They are found in abundance in nature, though not necessarily in
forms that organisms can use. Microorganisms mainly bacteria and fungi are primarily responsible for converting these
elements into usable forms. Many bacteria and fungi utilize organic molecules releasing carbon dioxide into the atmosphere,
which is used by algae, cyanobacteria, and higher plants to produce carbohydrates during photosynthesis. Bacteria also
convert atmospheric nitrogen into forms that can be used by plants and animals.
Microorganisms contribute to the production of various food products (bread, cheese, yogurt), beverages (alcohol, wine and
beer), medicines as antibiotics (e.g., penicillin, streptomycin, chloramphenicol), vaccines, vitamins, enzymes and many
other important products. Microbial products are widely used as aids to nutrition, prevention and treatment of diseases.

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Many microbial bio-products such as enzymes are used even for improving the quality of life. For example they are used in
drain cleaners to remove clogs without adding harmful chemicals to the environment, in detergent formulations and many
more.
On one hand when the vast majority of microbes benefit humans, animals, and plants in many ways, many microbial species
cause many fatal diseases also, however, fortunately only a minority of all microorganisms are pathogenic. A small group of
microbes cause food spoilage, such as soft spots on fruits and vegetables, decomposition of meats, and rancidity of fats and
oils.
Microorganisms have been heavily exploited for waste treatment and bioremediation over the last decade. They have been
used in sewage treatment, industrial effluent treatment, water recycling, etc. thereby preventing pollution of water bodies.
Bioremediation refers to use of microbes to clean up pollutants and toxic wastes produced by various industrial processes,
chemical spills, toxic waste sites, oil spills, etc. Microbes have also been used in control of insect pests in agriculture. Use of
microbes rather than chemical pesticides prevents the environment from harmful effects of chemical pesticides.
In some cases, microorganisms indigenous to the environment are used; in others, genetically modified microbes are also
used. Pseudomonas and Bacillusare the most commonly used bacteria that have been extensively studied and used for
different applications
The practical applications of microbiology to produce some common foods and chemicals contribute to biotechnology.
Some microorganisms are useful for mankind in their natural forms, while some may be genetically engineered to provide
the desired application. The use of recombinant DNA technology has contributed extensively to expand the potential of
bacteria, viruses, yeast cells and other fungi as miniature biochemical factories.
Points to remember

Microorganisms are helpful as well as harmful for other life forms.

They help to convert essential elements such as carbon and nitrogen to forms that can be utilized by plants and animals

Microorganisms are widely used in the food products, beverages, medicines as antibiotics, vaccines, vitamins, enzymes,
etc.

Microbial products are widely used as aids to nutrition, prevention and treatment of diseases.

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Microbiology in Food Industry

Beneficial & Detrimental Role of Microorganisms in Food Industry

Microorganisms play an important role in food industry. They are used in production of various food products, and are also
responsible for food spoilage thereby causing intoxication and diseases.
Microbial contamination of food products takes places usually on the way from the field to the processing plant, or during
processing, storage, transport and distribution or before consumption. The microorganisms that cause food spoilage and also
find the maximum exploitation in production of food and food products are mainly bacteria, molds and yeasts.
Bacteria
Bacteria are the largest group of unicellular microorganisms. The shapes of medically important bacteria are classified
into-cocci, or spherical cells; bacilli, or cylindrical or rod shaped cells; and spiral or curved forms. The pathogenic or
disease causing bacteria are usually gram negative, however, three gram-positive rods are known to cause food
intoxications : Clostridium botulinum,C. perfringens,and Bacillus cereus
Some of the other most common bacteria causing food spoilage, infections and disease areAcinetobacter, Aeromonas,
Escherichia, Proteus, Alcaligenes, Flavobacterium, Pseudomonas, Arcobacter, Salmonella, Lactococcus, Serratia,
Campylobacter, Shigella, Citrobacter, Listeria, Staphylococcus, Micrococcus, Corynebacterium, Vibrio Enterobacter,
Paenibacillus, Weissella, Enterococcus, Yersinia

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Different strains of bacteria are also used in production of various food and dairy products. Strains of
Streptococcus, Lactobacillus Bifidobacterium, Erwiniaetc. are used in the production of fermented food and dairy
products. Streptococcus thermophilus and Lactobacillusbulgaricus are used to produce yogurt.
Molds:
Molds are multicellular filamentous fungi whose growth on foods is usually readily recognized by their fuzzy or cottony
appearance. They are mainly responsible for food spoilage at room temperature 25- 30oC and low pH, and have minimum
moisture requirement. Molds can rapidly grow on grains and corns when these products are stored under moist conditions.
Molds require free oxygen for growth and hence grow on the surface of contaminated food.
Molds also find their use in manufacturing of different foods and food products. They are used in ripening of various types
of food products as cheese (e.g. Roquefort,Camembert). Molds are also grown as feed and food and are employed to
produce ingredients such as enzymes like amylase used in making bread or citric acid used in soft drinks. Molds are major
contributors in the ripening of many oriental foods. A species of Bothrytiscinerea, is used in rotting of grape for production
of wine. Lactic fermentations using molds results in a unique Finnish fermented milk called viili.
Yeasts:
Yeasts have the ability to ferment sugars to ethanol and carbon-dioxide and hence they are extensively in food industry. The
most commonly used yeast, the bakers yeast is grown industrially. Saccharomyces carlsbergensis is most commonly used
in fermentation of most beers. The other yeast strains of importance are
Brettanomyces, Schizosaccharomyce,, Candida, Cryptococcus, Debaryomyces, Zygosaccharomyces,
Hanseniaspora, Saccharomyces
Food Microbiology covers studies on:

Food spoilage by different kinds of microorganisms such as bacteria and fungi

Testing of Food/ food products for microbial contamination

Methods to be employed for prevention of food spoilage and preservation techniques

Use of various microorganisms in production of various food products

Microorganism Growth and Food Spoilage

Different food products provide ideal growth conditions for microorganisms. Microbial growth is controlled by intrinsic
factors like nutrients, pH, moisture content, physical structure of the food and/ or extrinsic factors like temperature, relative

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humidity, gases (CO2,O2). Microorganisms thus grow in optimum conditions provided by external and internal factors,
resulting in spoilage and degradation of the food product resulting in a sour, foul-smelling or fungus-covered inedible mass.
Microbial growth in foods can also cause visible changes like change in colour, deposition of powdery growth,
effervescences on food surface, etc. Microbial contamination of food can occur at any point in the food production process:
growth, harvesting, transport, storage, or final preparation. Spoilage also can occur if foods are not stored properly. Meat
and dairy products are rich in protein and fat serves as an ideal environment for microbial spoilage resulting in proteolysis
and putrefaction of the food products. Vegetables and fruits have a much lower protein and fat content and undergo a
different kind of spoilage.
Microbiological testing of Food
There are basically two main strategies for testing food products.
1.

Determination of Microbiological Quality: This is done by determining total viable count of the food sample. This
includes Total bacteria count (TBC), Total yeast and mold count (TYMC). This test provides the details about
microbial quality of the food

2.

Determination of Food Safety: This test includes analysis and testing of food samples for food pathogens belonging to
group Enterobacteria, Enterococci, coliforms, E. coli and any other pathogens as Pseudomonas sp. Clostridia,
Salmonella, Bacillus spp, Staphylococcus, lactic acid bacteria, Salmonella, Listeria, yeasts and molds etc. .

Control of Microbial Growth and Food Preservation

Contamination of food may occur due to the presence of any microbial population during packaging, after opening the
packaged food or serving of the food. To ensure long shelf life of preserved and stored food it is vital to eliminate or reduce
the populations of spoilage and disease-causing microorganisms and to maintain the microbiological quality of a food with
proper storage and packaging. Microbial growth in food products can be controlled by various methods.

Removal of microorganisms by physical methods such as filtration, centrifugation

Thermal treatment as Refrigeration, freezing, partial or complete heat inactivation of microorganisms (pasteurization
and canning)

Reduction or removal of moisture/water content by freeze drying (lyophilization)

Addition of chemical preservatives

Use of ionizing (gamma rays) and non ionizing (UV) radiation

Addition of substances such as bacteriocins based inhibition to foods to control food-borne pathogens

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Microorganisms in Food Production

1.

Microorganisms have been widely used in lactic, propionic, and ethanolic fermentations from time immemorial.

2.

They are characteristically used in production of about 2,000 distinct varieties of cheese throughout the world.

3.

They are used in production of different alcoholic beverages using variety of carbohydrate substrates.

4.

Microorganisms can be used to transform raw foods into pickles, sausages, sauces,etc.

5.

Microorganisms can themselves be used as a food source. The most common example is fungus (Agaricusbisporus)
commonly as mushrooms.

6.

Probiotics are being used successfully with poultry.

Points to remember

Most food and food products provide an excellent environment for microbial growth leading to food spoilage and
degradation.

Various intrinsic and extrinsic factors as pH, sugar and salt content of the food, moisture content, temperature etc. are
responsible for the growth of microorganisms in a food product.

Microorganisms can spoil meat, dairy products, fruits, vegetables, and canned goods in several ways.

Microbial testing of food involves the quality and safety analysis of the food samples.

Microbial contamination of food can be prevented by various methods as physical removal of microorganisms, thermal
treatment, freeze drying, addition of chemicals, radiation etc.

Microorganisms are used in the production of various fermented products, cheese, sauces, pickles, alcoholic beverages
etc.Bacteria, molds and yeast are the most important microorganisms that cause food spoilage and also find the
maximum exploitation in production of food and food products.

Different strains of bacteria and fungus are used for fermentation of dairy products for production of a wide variety of
cultured milk products. Both bacteria and fungi are used in these cheese production processes.

Lactic acid bacteria are used for coagulation of milk that can be processed to yield a wide variety of cheeses, including
soft unripened, soft ripened, semisoft, hard, and very hard types.

Microorganisms such as Lactobacillus and Bifidobacterium are used as in food and health industry.

Spirulina, a cyanobacterium, also is a popular food source sold in specialty stores.

Molds are used for rotting of grapes for production of different varieties of wines.

Mushrooms (Agaricusbisporus) are one of the most important fungi used as a food source.

Alcoholic beverages as beer are produced by fermentation of cereals and grains using different strains of yeasts.

Bacteria, molds and yeast are the most important microorganisms that cause food spoilage and also find the maximum
exploitation in production of food and food products.

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Different strains of bacteria and fungus are used for fermentation of dairy products for production of a wide variety of
cultured milk products. Both bacteria and fungi are used in these cheese production processes.

Lactic acid bacteria are used for coagulation of milk that can be processed to yield a wide variety of cheeses, including
soft unripened, soft ripened, semisoft, hard, and very hard types.

Microorganisms such as Lactobacillus and Bifidobacterium are used as in food and health industry.

Spirulina, a cyanobacterium, also is a popular food source sold in specialty stores.

Molds are used for rotting of grapes for production of different varieties of wines.

Mushrooms (Agaricusbisporus) are one of the most important fungi used as a food source.

Alcoholic beverages as beer are produced by fermentation of cereals and grains using different strains of yeasts.

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Microbiology in Pharmaceutical Industry

Contributions of Microbiology to Pharmaceutical Industry

The most important contribution of microbiology to the pharmaceutical industry is the development of antibiotics. All
antibiotics were originally the products of microbial metabolism, however the recent genetic manipulations have enabled the
production of more enhanced drugs. Vaccines are also a very important contribution of microbiology towards development
of drugs. The production of vaccines against bacterial diseases usually requires the growth of large amounts of bacteria.
Steroids can also be obtained from microorganisms.
Apart from drugs and bio products development, microbiology contributes towards quality control of a pharmaceutical
laboratory. Prevention of microbial contamination of drugs, injectables, eye drops, nasal solutions and inhalation products is
undertaken following pharmacopeial guidelines.
Microbiological Test Methods
Microbiological tests for pharmaceuticals fall into several categories.
The Growth Promotion test
The growth promotion test is an important quality control function in the pharmaceutical industry. It is imperative for
establishing the ability and nutritive property of any media used to support growth when the inoculum contains a small
number of microorganisms.

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Sterility Testing.
Sterility testing is done on wide range of pharmaceutical products as parental preparations, ophthalmic & other
non-injectable preparations, bulk solids and liquid solutions, antibiotic solids, and medical consumables and devices.
Microbial Limits Test
This test is used to estimate the total number of viable microorganisms or specific pathogens present in pharmaceutical
products as tablets, capsules, oral suspensions, injectables, ophthalmic and nasal solutions and other medical devices. It is
based on the principle that any viable microbial cell present in a sample will produce a single colony when provided with a
growth medium and favourable growth conditions. The enumeration of these colony-forming units (cfu) gives an estimate
of the microbial population of the product. The microbial content of the product includes the total bacterial count (TBC),
total yeast and mold count (TYMC). These tests are mandatory for the release of drug products.
Bioburden Testing
Bioburden of raw materials and finished pharmaceutical products helps to determine whether the product complies with the
requirements of the US Pharmacopeia. Bioburden is the total number of microorganisms present on a product prior to
sterilisation.
Water Testing
Water is one of the major commodities consumed by the pharmaceutical industry. Total viable count is studied to rule out
microbial contamination. Tests for presence of coliforms, E. coli and any other pathogens
as Pseudomonas sp. Clostridia, Salmonella, Staphylococcus etc.are performed.
Bacterial Endotoxin (LAL Testing)
Endotoxins are natural compounds released by the cell wall of gram negative bacteria that are potentially toxic to humans.
This material is pyrogenic (causing high fevers in humans). The test for bacterial Endotoxin is used to detect or quantify
endotoxins using Limulus Amoebocyte Lysate (LAL) which is an extract of blood cells from the horseshoe crab (Limulus
polyphemus)
Drug quality and safety is the most important aspect of microbiological testing pharmaceutical products. The presence of
any pathogenic bacteria, yeasts, moulds or bacterial toxins produced by microorganisms is strictly regulated to ensure
prevention of any risk.

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Points to remember

Microorganisms are used in the production of antibiotics, vaccines, steroids, etc

Microbial testing of pharmaceutical products is strictly followed as per pharmacopeial guidelines.

Growth promotion tests establish the potential of any media to support growth when the inoculum contains a small
number of microorganisms.

Microbial limit testing and sterility testing are used to identify the microbial load of the product.

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Common Interview Questions for Microbiologist

with Answers

Job Interview

1.What is the difference between procaryotic and eukaryotic cells?

Ans. Prokaryotic cells differ from eucaryotic cells in lacking a membrane bound nucleus and other cell organelles. They are
much smaller in size (Typically 0.2-2.0 mm in diameter) compared to the eukaryotic cells (Typically 10-100 mm in
diameter). Prokaryotic cells usually have a complex cell wall while eukaryotic cells do not have a cell wall, or have a very
simple one.
2.What magnification is required to observe microorganisms?
Ans. Basic magnification required to view and focus on microorganisms is 40x. Larger cells can be seen clearly, but the
more intricate details remain invisible. At 100x bacteria are visible as small dots with little details. 400x magnification is
necessary for studying minute details.
3.What microbial characteristics should be determined during examination and investigation of microorganisms?
Ans. The microorganisms should be studied on the basis of morphological characteristics, chemical composition, cultural,
metabolic, genetic characteristics, antigenic properties, pathogenicity, and ecological characteristics

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4.Why bacteria require staining before microscopic examination?

Ans. Bacteria have nearly the same refractive index as water, which makes them visually opaque even when observed under
a microscope. Hence, microorganisms are routinely stained to make them more visible under the light microscope. The cells
must be fixed and stained to increase visibility, accentuate specific morphological features and preserve them for subsequent
use. Different types of staining techniques are required. Staining is thus an auxiliary technique used in microscopy and
microbiology to enhance contrast in the microscopic images of the microorganisms.
5.Why is gram stain one of the most important and widely used stains in bacteriology?
Ans. The Gram stain is generally the first step in the identification of a bacterial organism. It is a valuable diagnostic tool in
both clinical and research settings, Gram staining differentiates bacteria by the chemical and physical properties of their cell
walls that contain peptidoglycan, which is present as a thick layer in gram-positive bacteria, while as a thin layer in the gram
negative bacteria.
6.What are endospores. Can it be referred to as the method of reproduction?
Ans. Endospores are thick walled highly refractile bodies that are produced one per cell by some microorganisms as
Bacillus, Clostridium, Sporosarcina, Thermoactinomyces. They are extremely resistant to desiccation, staining, disinfection
and many sterilization processes. Spore is a metabolically dormant form which under suitable conditions, can undergo
germination and form vegetative cell. However it is not a method of reproduction.
7.Why is nutrient broth considered as a universal growth medium for bacteria?
Ans. Nutrient broth is considered a universal growth medium because it contains ingredients such as beef extract, peptone,
and yeast extract that provide the ingredients such as carbohydrates, organic nitrogen compounds, water soluble vitamins
and salts
8.What is the use of pure cultures?
Ans. Pure cultures are needed for laboratory and research work as test agents for various studies or as reference strains for
taxonomic studies. They enable the scientists to study the characteristics of a single species.
9.What is the most common sterilization technique used in laboratories. Explain the technique?

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Ans. The most common method of sterilization used in the laboratories is the use of autoclave, which works on the principle
of destruction of microorganisms by employing moist heat. It uses high pressure and temperature (121oC at 15 psi for 15
min), for killing the vegetative and spore forms of the microbial cells
10.What is the scope of microbiology?

Ans. Microorganisms affect the well being of humans in a lot of ways. They degrade dead plants and animals
and recycle chemical elements to be used by living plants and animals. They are used to decompose organic
matter in sewage and other wastes (as in bioremediation). They are used as biopestcides, in production of food
products,enzymes, drugs, medicines, vaccines. In gene therapy, viruses are used to carry replacements for
defective or missing genes into human cells. Genetically modified bacteria are used in agriculture to protect
plants from frost and insects and to improve the shelf life of produce.

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Chapter-13: Conclusions
We believe that you enjoyed the free e- course on Microbiology.The last chapter of the course provides answers to 10
common questions that you may be faced with as you move up your career ladder. However, learning is a lifelong process
and there will be several unanswered questions and queries which will be coming up in your mind from time to time. Our
suggestion to you would be to post such queries or comments on the site and we shall try to offer clarifications to the best of
our ability based on our expertise and experience.

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