Microbiology e Book

7/21/2019 Microbiology e Book

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By

Dr. Saurabh Arora

Founder : Lab-Training.com

Director : Auriga Research Ltd., Arbro Pharmaceuticals Ltd. (Analytical Division)

E-mail : [email protected]

&

Dr. Rajshree Saxena

Auriga Research Ltd.

And

Arbro Pharmaceuticals Ltd.

Analytical Division,

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

mailto:[email protected]

mailto:[email protected]



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Author’s 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 team’s 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.

http://www.lab-training.com/

http://www.foodsafetyhelpline.com/

http://www.foodsafetyhelpline.com/

http://www.lab-training.com/



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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.



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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.



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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



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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 μm, they lack a

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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 μm and yeast has size varying in the range of 5-10 μm.

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.



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Observing Microorganisms-Microscopic

ExaminationMicrobiology 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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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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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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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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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 β 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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General Classification and Characterization of

MicroorganismsClassification 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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The taxonomic classification scheme for prokaryotes is elaborated in Bergey’s 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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Bergey’s Manual provides classification scheme for bacteria as well as a reference for identification of different bacteria in

the laboratory. According to Bergey’s 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 Bergey’s, 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 Bergey’s Manual of Systematic Bacteriology

according to which the prokaryotes are divided into two domains: Bacteria and Archaea



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Prokaryotic Cell-Brief description of Structure and

functionProkaryotic cells fall into a size range of about 1–5µm 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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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 cell’s 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 cell’s 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 isn’t 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

present

ER, golgi body, lysosomes,

mitochondria, lysosomes

Nucleus Not well defined, without any nuclear

membrane or nucleoli

Well defined nucleus present, with

nuclear membrane and nucleus

Chromosome Single circular chromosome present as

nuclear material without histones

Multiple linear chromosomes found

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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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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Conditions Favourable to Growth of

Microorganisms

Microorganisms

All living organisms, microorganisms require a combination of various physical and chemical factors for their growth andmultiplication. 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 organic 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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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

Photoorganoheterotrophy Light Energy Purple nonsulphur bacteria

Organic hydrogen/electron

donor Green nonsulphur

Organic carbon source

Chemolithoautotrophy Chemical (organic) energy

source Sulphur oxidising bacteria

Inorganic hydrogen/electron

donor Hydrogen bacteria

Carbon source- Nitrifying, Iron oxidising

bacteria

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

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

organotrophs



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Cultures-the Growth and Survival Media of

MicroorganismsThe artificial 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 121°C 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 37°C for bacteria and 25-28°C for yeast and molds). The petri plates with bacterial inoculation are keptin 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 37°C and 25-28°C 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 baker’s 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 indiameter). 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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