Basic Microbiology(2101)

Module-1
History and Scope of Microbiology
➢ Overview of history of Microbiology:
Biogenesis
Biogenesis is the scientific principle that life arises from pre-existing life, not from non-
living material. This concept is supported by significant experimental evidence and is a
cornerstone of modern biology. The idea was famously supported by Louis Pasteur's
experiments in the 19th century, which demonstrated that microorganisms in nutrient broth
came from other microorganisms in the air, not spontaneously from the broth itself. This
principle essentially states that all living organisms are derived from other living
organisms, maintaining a continuous line of life.

Abiogenesis
Abiogenesis is the hypothesis that life can arise from non-living matter through natural
processes. This concept pertains to the origin of life on Earth. It suggests that simple
organic compounds could have given rise to more complex molecules, eventually leading
to the first primitive life forms. While abiogenesis is a widely studied field, it remains a
hypothesis since direct observational evidence is challenging to obtain. Various theories
and experiments, such as the Miller-Urey experiment, have shown that organic molecules
necessary for life can form under prebiotic conditions, supporting the plausibility of
abiogenesis.
➢ Francesco Redi (1626-1697)
• Contribution: Redi was one of the first to challenge the concept of spontaneous
generation.
• Experiment: In 1668, he demonstrated that maggots on decaying meat came from fly
eggs, not spontaneously from the meat itself. He covered some jars with gauze to
prevent flies from laying eggs and observed that no maggots appeared in the covered
jars, while they did in the open jars.
➢ Lazzaro Spallanzani (1729-1799)
• Contribution: Spallanzani conducted experiments to further disprove spontaneous
generation.
• Experiment: He boiled nutrient broths and sealed some containers while leaving others
open. The sealed containers remained free of microbial life, suggesting that
microorganisms came from the air and not spontaneously from the broth.
➢ John Needham (1713-1781)
• Contribution: Needham initially supported spontaneous generation.
• Experiment: He boiled broth, sealed it, and still observed microbial growth,
concluding that life could spontaneously arise. However, his experiments were later
criticized for not being properly sealed and for insufficient boiling time.
➢ Louis Pasteur (1822-1895)
• Contribution: Pasteur conclusively disproved spontaneous generation and contributed
to the development of the germ theory of disease.

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 • Experiment: In his famous swan-neck flask experiments, he showed that sterilized
broth remained free of microorganisms unless exposed to air containing particles of
dust, which carried microorganisms. He also developed pasteurization and vaccines for
rabies and anthrax.
➢ John Tyndall (1820-1893)
• Contribution: Tyndall provided evidence that supported Pasteur’s findings.
• Experiment: He demonstrated that certain broths remained sterile if not exposed to
dust, proving that microorganisms in the air could contaminate broths. He also
discovered the existence of heat-resistant bacterial spores.
➢ Joseph Lister (1827-1912)
• Contribution: Lister pioneered antiseptic surgery.
• Development: He introduced the use of carbolic acid (phenol) to sterilize surgical
instruments and clean wounds, significantly reducing post-operative infections and
mortality.
➢ Robert Koch (1843-1910)
• Contribution: Koch is one of the founders of bacteriology and formulated Koch's
postulates.
• Achievements: He identified the causative agents of tuberculosis, cholera, and anthrax.
His postulates are criteria for establishing a causal relationship between a microbe and
a disease.
➢ Edward Jenner (1749-1823)
• Contribution: Jenner is known as the father of immunology.
• Achievement: He developed the smallpox vaccine, the world's first vaccine. By
inoculating a boy with cowpox virus and demonstrating immunity to smallpox, Jenner
laid the foundation for modern vaccination practices.
➢ Alexander Fleming (1881-1955)
• Contribution: Fleming discovered penicillin, the first true antibiotic.
• Discovery: In 1928, he observed that the mold Penicillium notatum produced a
substance that killed a wide range of bacteria. This discovery led to the development of
antibiotics, revolutionizing the treatment of bacterial infections.
Scope of Microbiology.
Microbiology is a vast and dynamic field encompassing the study of microorganisms,
including bacteria, viruses, fungi, protozoa, and algae. The scope of microbiology is broad
and can be categorized into various sub-disciplines and applications:
➢ Sub-disciplines of Microbiology

1) Bacteriology: Study of bacteria, including their classification, physiology, and role in

disease and ecology.

2) Virology: Study of viruses and virus-like agents, including their structure, classification,
and the diseases they cause.

3) Mycology: Study of fungi, including yeasts and molds, focusing on their biology, genetics,
and role in health and disease.

4) Parasitology: Study of parasites, including protozoa and helminths, focusing on their life
cycles, host interactions, and diseases they cause.

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5) Phycology (Algology): Study of algae, including their classification, ecology, and use in
biotechnology.

6) Microbial Genetics: Study of how microorganisms inherit traits and how their genetic
material is structured, functioned, and manipulated.

7) Microbial Ecology: Study of the interactions of microorganisms with their environment,

including their roles in nutrient cycling, biodegradation, and ecosystems.

8) Industrial Microbiology: Application of microbial sciences to industrial processes,

including the production of biofuels, enzymes, and pharmaceuticals.

9) Food Microbiology: Study of microorganisms in food, including their roles in

fermentation, spoilage, and foodborne diseases.

10) Medical Microbiology: Study of microorganisms that cause diseases in humans and
animals, including the development of diagnostic methods, treatments, and vaccines.

11) Environmental Microbiology: Study of microbial processes in the environment,

including soil, water, and air microbiology.
➢ Applications of Microbiology

1) Healthcare and Medicine:

• Disease Diagnosis: Identification of pathogens causing infections.
• Vaccine Development: Creating vaccines to prevent infectious diseases.
• Antibiotic Production: Developing antibiotics to treat bacterial infections.
• Antiviral Therapies: Developing treatments for viral infections.
• Immunotherapy: Using microorganisms or their products to modulate immune
responses.

2) Agriculture:
• Soil Fertility: Studying soil microbes that enhance nutrient availability.
• Biopesticides: Developing microbial agents to control agricultural pests.
• Plant Growth Promotion: Using beneficial microbes to enhance crop yields.

3) Food Industry:
• Fermentation: Producing fermented foods and beverages like yogurt, cheese, beer, and
wine.
• Food Safety: Ensuring food products are free from harmful microorganisms.

4) Biotechnology:
• Genetic Engineering: Using microbes for recombinant DNA technology.
• Biofuels: Producing renewable energy sources like ethanol and biodiesel.
• Bioremediation: Using microbes to clean up environmental pollutants.

5) Environmental Science:
• Waste Treatment: Using microbes in sewage and waste treatment processes.
• Pollution Control: Monitoring and mitigating microbial impacts on air and water
quality.
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 • Climate Change: Studying microbial roles in carbon and nitrogen cycling.

6) Pharmaceutical Industry:
• Drug Discovery: Exploring microbial products for new drugs and therapies.
• Probiotics: Developing microbial products to promote gut health.

7) Research and Development:

•Fundamental Research: Understanding basic microbial physiology, genetics, and
ecology.
• Applied Research: Developing new technologies and applications based on microbial
properties.
➢ Emerging Areas

1) Microbiome Research: Studying the collective genomes of the microorganisms in a

particular environment, particularly the human microbiome and its impact on health.

2) Synthetic Biology: Engineering new microorganisms with desirable traits for industrial,
medical, and environmental applications.

3) Astrobiology: Studying the potential for microbial life beyond Earth.

4) Microbial Forensics: Using microbial signatures to solve crimes and track sources of
outbreaks.

Systems of classification:
The classification of organisms has evolved over time to reflect our increasing
understanding of the diversity and relationships among living things. Two influential
systems are Robert H. Whittaker's five-kingdom classification and Carl Woese's three-
domain system.

➢ Whittaker’s Five Kingdom Classification (1969)

Robert H. Whittaker proposed a
five-kingdom system to
categorize all life forms based on
their cellular organization and
modes of nutrition. The five
kingdoms are:

1) Monera:
• Organisms: Prokaryotes,
including bacteria and
cyanobacteria.
• Characteristics: Single-
celled, lack a nucleus, have a
simple cell structure.

2) Protista:
• Organisms: Mostly single-celled eukaryotes, such as amoebas, algae, and protozoa.
• Characteristics: Have a nucleus, can be autotrophic or heterotrophic.
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3) Fungi:
• Organisms: Molds, yeasts, mushrooms.
• Characteristics: Eukaryotic, primarily multicellular (except yeasts), heterotrophic,
absorb nutrients from their environment.

4) Plantae:
• Organisms: Plants, including mosses, ferns, conifers, and flowering plants.
• Characteristics: Multicellular, eukaryotic, autotrophic (photosynthetic).

5) Animalia:
• Organisms: Animals, including sponges, insects, fish, birds, and mammals.
• Characteristics: Multicellular, eukaryotic, heterotrophic, ingest food.
Utility: Whittaker's system was revolutionary in distinguishing between prokaryotic and
eukaryotic organisms and recognizing fundamental differences in nutrition (autotrophy vs.
heterotrophy). It provided a more comprehensive framework than previous two- or three-
kingdom systems.

➢ Carl Woese’s Three-Domain Classification (1990)

Carl Woese introduced a three-
domain system based on
differences in ribosomal RNA
(rRNA) sequences, recognizing
the deep evolutionary
relationships among life forms.
The three domains are:

1) Bacteria:
• Organisms: True bacteria
(prokaryotes).
• Characteristics: Single-
celled, lack a nucleus, have peptidoglycan in cell walls.

2) Archaea:
• Organisms: Archaebacteria, which include extremophiles.
• Characteristics: Single-celled, prokaryotic, lack peptidoglycan, have unique
membrane lipids, and different rRNA sequences compared to bacteria.

3) Eukarya:
• Organisms: All eukaryotic organisms, including protists, fungi, plants, and animals.
• Characteristics: Cells with a true nucleus and membrane-bound organelles.
Utility: Woese's system highlights the genetic and evolutionary distinctions between the
traditional prokaryotes, separating them into Bacteria and Archaea. This classification
underscores the importance of molecular data in understanding evolutionary relationships
and the profound differences between these two groups of prokaryotes.
➢ Comparison and Utility
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1) Whittaker’s System:
• Strengths: Simple and intuitive, distinguishes major life forms based on cell type,
complexity, and nutrition.
• Limitations: Lumps all prokaryotes into one kingdom (Monera), does not reflect
genetic and evolutionary relationships as accurately as later systems.

2) Woese’s System:
• Strengths: Based on molecular data (rRNA sequences), reflects evolutionary
relationships more accurately, particularly among prokaryotes.
• Limitations: More complex, not as intuitive for distinguishing broader life forms
without understanding molecular genetics.
Classification of Microbes (Microbial Taxonomy
Microbial taxonomy is the classification of microorganisms based on a variety of
characteristics, ranging from morphological and physiological traits to genetic and
molecular data. Here are the key identifying characters used in the classification of
microbes:
➢ Morphological Characteristics

1) Cell Shape:
• Bacteria: Cocci (spherical), Bacilli (rod-shaped), Spirilla (spiral-shaped), Vibrios
(comma-shaped), Spirochetes (corkscrew-shaped).
• Fungi: Yeasts (unicellular, oval-shaped), Molds (filamentous, forming hyphae),
Dimorphic fungi (exist as both yeast and mold forms).

2) Cell Size: Variations in size are used to distinguish between different microbial species and
groups.

3) Cell Arrangement:
• Bacteria: Diplococci (pairs), Streptococci (chains), Staphylococci (clusters), Tetrads
(groups of four), Sarcinae (cubical packets).
• Algae and Protozoa: Unicellular or multicellular arrangements, colony formation.

4) Motility: Presence and type of locomotion structures such as flagella, cilia, or pseudopodia.
5) Cell Wall Structure:
• Bacteria: Gram-positive (thick peptidoglycan layer) or Gram-negative (thin
peptidoglycan layer and outer membrane).
• Fungi: Presence of chitin in the cell wall.
• Archaea: Unique cell wall composition without peptidoglycan.

6) Spore Formation: Production of endospores (e.g., Bacillus, Clostridium) or reproductive

spores (e.g., fungal spores).

7) Colony Morphology: Appearance of microbial colonies on solid media, including shape,

size, color, texture, and margin.
➢ Physiological and Metabolic Characteristics

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1) Oxygen Requirements: Aerobes (require oxygen), Anaerobes (do not require oxygen),
Facultative anaerobes (can grow with or without oxygen), Microaerophiles (require low
oxygen levels).

2) Temperature Preferences: Psychrophiles (cold-loving), Mesophiles (moderate

temperatures), Thermophiles (heat-loving), Hyperthermophiles (extremely high
temperatures).

3) pH Tolerance: Acidophiles (acidic environments), Neutrophiles (neutral pH), Alkaliphiles

(alkaline environments).

4) Nutritional Requirements: Autotrophs (use CO2 as a carbon source), Heterotrophs (use

organic compounds), Mixotrophs (capable of both autotrophy and heterotrophy).

5) Metabolic Pathways: Types of respiration (aerobic, anaerobic), fermentation processes,

photosynthesis mechanisms (oxygenic or anoxygenic).

6) Enzyme Activity: Presence of specific enzymes like catalase, oxidase, urease, and their
activity in biochemical pathways.
➢ Biochemical Characteristics

1) Carbohydrate Fermentation: Ability to ferment specific sugars and produce gas or acid
(e.g., lactose, glucose).

2) Protein and Amino Acid Utilization: Ability to deaminate or decarboxylate amino acids,
production of indole, hydrogen sulfide.

3) Lipid Utilization: Production of lipases and other enzymes involved in lipid degradation.
4) Production of Secondary Metabolites: Antibiotics, pigments, toxins, and other bioactive
compounds.

5) Gas Production: Production of gases like hydrogen, methane, and hydrogen sulfide during
metabolic processes.
➢ Genetic and Molecular Characteristics

1) DNA Sequencing: Analysis of specific genes such as 16S rRNA for bacteria, ITS regions
for fungi, and other conserved genetic markers.

2) Genomic G+C Content: The percentage of guanine and cytosine bases in the DNA, used
to distinguish different groups of microorganisms.

3) Hybridization Techniques: DNA-DNA hybridization to determine genetic similarity and

relatedness.

4) Polymerase Chain Reaction (PCR): Amplification and analysis of specific genetic

sequences.

5) Ribotyping: Comparison of ribosomal RNA gene restriction patterns for classification and
identification.

6) Multilocus Sequence Typing (MLST): Sequencing multiple housekeeping genes to

identify strains and species.

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➢ Serological Characteristics

1) Antigenic Properties: Reaction with specific antibodies, used in serotyping to differentiate

strains and species based on their antigenic makeup.

2) Agglutination Tests: Clumping of cells in the presence of specific antibodies, indicating

the presence of particular antigens.
➢ Ecological Characteristics

1) Habitat: Specific environments where the microorganism is found, such as soil, water,
human body, extreme environments.

2) Symbiotic Relationships: Types of interactions with other organisms, such as mutualism,

commensalism, parasitism.

3) Pathogenicity: Ability to cause disease in a host organism, virulence factors, host range.
➢ Chemotaxonomic Characteristics

1) Cell Wall Components: Composition of the cell wall, such as peptidoglycan types in
bacteria, chitin in fungi.

2) Membrane Lipids: Types of lipids present in the cell membrane, such as fatty acid profiles,
phospholipids, sterols.

3) Quinones: Types of quinones present in the electron transport chain, such as ubiquinones
and menaquinones.
➢ Microbial taxonomy
Microbial taxonomy involves the classification, identification, and nomenclature of
microorganisms. This field aims to organize microorganisms into groups based on shared
characteristics, providing a framework for understanding their relationships and roles in
various environments. Here are the general properties and principles of microbial
classification, along with details on bacterial systematics, numerical taxonomy, and the
general properties of Archaea and Eubacteria.
General Properties and Principles of Classification of Microorganisms

1) Morphological Characteristics: Shape, size, and arrangement of cells. Presence and

structure of cell walls, flagella, pili, and spores.

2) Physiological and Metabolic Traits: Growth requirements such as oxygen, temperature,

pH, and nutrient preferences. Metabolic capabilities including fermentation, respiration,
and photosynthesis.

3) Biochemical Properties: Enzymatic activities, utilization of specific substrates, and

production of metabolic end-products. Reactions to various biochemical tests (e.g.,
catalase, oxidase tests).

4) Genetic and Molecular Data: DNA/RNA sequencing, especially 16S rRNA gene for
bacteria. G+C content, DNA-DNA hybridization, and multilocus sequence typing (MLST).

5) Ecological Characteristics: Natural habitats, ecological roles, symbiotic relationships, and

pathogenicity.
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6) Chemotaxonomic Features: Cell wall components, membrane lipids, and quinones.
Specific biochemical markers and secondary metabolites.

7) Serological Properties: Antigenic properties detected by specific antibodies. Serotyping

methods like agglutination and ELISA.
Systematics of Bacteria
Bacterial systematics involves the study of the diversity of bacterial species and their
evolutionary relationships. The main methods and criteria include:

1) Phenotypic Methods:
• Morphology: Microscopic examination of cell shape, size, and structure.
• Physiology: Growth conditions, metabolic activities, and biochemical tests.
• Serology: Antigen-antibody interactions.

2) Genotypic Methods: 16S rRNA sequencing: Comparing sequences to identify and classify
bacteria. Whole-genome sequencing: Provides comprehensive genetic information. DNA-
DNA hybridization: Measures genetic similarity between organisms.

3) Phylogenetic Analysis: Constructing phylogenetic trees based on genetic data to show

evolutionary relationships.
Numerical Taxonomy
Numerical taxonomy (or phenetics) involves the classification of organisms based on a
large number of characteristics, each given equal weight. The process includes:

1) Data Collection: Gather extensive phenotypic data on morphological, physiological, and

biochemical traits.

2) Similarity Coefficients: Calculate similarity coefficients (e.g., Jaccard, Sneath, and Sokal
coefficients) to quantify the degree of similarity between organisms.

3) Cluster Analysis: Use algorithms (e.g., UPGMA, Ward’s method) to group organisms into
clusters based on their overall similarity.

4) Dendrograms: Create dendrograms (tree-like diagrams) to visually represent the

relationships and classification of the organisms.

General Properties of Archaea and Eubacteria

Archaea and Eubacteria are the two primary domains of prokaryotic life, each with
distinct properties:
➢ Archaea

1) Cell Structure: Lack peptidoglycan in their cell walls; instead, they have
pseudopeptidoglycan or other unique polymers. Membrane lipids consist of ether-linked
isoprenoids.

2) Genetics: Unique rRNA sequences distinct from Eubacteria. Similarities to eukaryotes in

transcription and translation machinery.

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3) Metabolism: Capable of surviving in extreme environments (extremophiles), such as high
temperatures (thermophiles), high salinity (halophiles), or acidic conditions (acidophiles).
Unique metabolic pathways, such as methanogenesis.

4) Ecology: Found in diverse environments, including extreme habitats and more common
ones like soil and water. Play key roles in nutrient cycles, such as carbon and nitrogen
cycling.
➢ Eubacteria (Bacteria)

1) Cell Structure: Cell walls typically contain peptidoglycan (Gram-positive and Gram-
negative types). Membrane lipids are ester-linked fatty acids.

2) Genetics: Distinct rRNA sequences from Archaea and eukaryotes. Simple transcription and
translation mechanisms compared to eukaryotes.

3) Metabolism: Extremely diverse metabolic capabilities, including aerobic and anaerobic

respiration, fermentation, photosynthesis, and nitrogen fixation.

4) Ecology: Ubiquitous in all environments (soil, water, air, living organisms). Involved in
essential processes such as decomposition, nutrient cycling, and symbiotic relationships
(mutualism, commensalism, parasitism).

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 Module-2
Stains and Staining Techniques
➢ Definitions of Auxochrome, Chromophore, and Dye
Auxochrome
• Definition: An auxochrome is a functional group attached to a chromophore that
modifies the ability of the chromophore to absorb light. Auxochromes do not
themselves cause a color change but enhance the color and solubility of the dye.
• Examples: Common auxochromes include hydroxyl groups (-OH), amino groups (-
NH2), and carboxyl groups (-COOH).
• Function: By altering the electron distribution in the chromophore, auxochromes can
shift the wavelength of maximum absorption, often intensifying the color (a process
known as bathochromic shift) and sometimes changing the hue.
Chromophore
• Definition: A chromophore is the part of a molecule responsible for its color. It is a
region within the molecule where the energy difference between two molecular orbitals
falls within the visible spectrum, allowing the molecule to absorb certain wavelengths
of light and thus appear colored.
• Examples: Conjugated systems with alternating double and single bonds, such as the
azo group (-N=N-) in azo dyes, the nitro group (-NO2), and carbonyl groups (C=O).
• Function: When light is absorbed by the chromophore, an electron is excited from a
lower energy molecular orbital to a higher energy molecular orbital. The specific
wavelengths absorbed and thus the color seen depend on the structure of the
chromophore.
Dye
• Definition: A dye is a colored substance that has an affinity for the substrate to which it is
being applied. Dyes are typically used in solutions and have the ability to adhere to or
interact with materials like textiles, paper, leather, or biological tissues.
• Types:
1) Acid Dyes: Water-soluble and used on fibers such as wool and silk.
2) Basic Dyes: Usually cationic and used for acrylic fibers and in histology.
3) Direct Dyes: Applied directly to the substrate in an aqueous solution without the need
for a mordant.
4) Mordant Dyes: Require a binding agent (mordant) to fix the dye to the substrate.
5) Reactive Dyes: Form a covalent bond with the substrate.
6) Disperse Dyes: Mainly used for synthetic fibers.
• Function: The primary role of dyes is to impart color to materials by forming bonds with
them, either physically or chemically.
Relationship Between Auxochrome, Chromophore, and Dye
In a dye molecule, the chromophore is responsible for the absorption of light and the
resultant color. The auxochrome, when attached to the chromophore, modifies the ability
of the chromophore to absorb light, enhancing or altering the visible color. Together, the

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 chromophore and auxochrome form the structure of the dye, determining its color
properties and suitability for different applications.

Staining
Staining is a method of imparting colour to cells, tissues or microscopic components, so
they are highlighted and visualized better under a microscope. There are a variety of
staining methods like simple, differential and special staining, which are used for various
purposes ranging from the study of microscopic organisms to cellular structures, metabolic
processes, cytopathology to name a few.
Staining is carried out with the help of a reagent termed as “stain“. This method uses a wide
variety of natural and synthetic stains, which is used to add colour to the colourless
specimen to be studied. It can be done in two ways, namely in-vitro and in-vivo, that is
explained below:

The protocol of staining generally involves three sequential stages:

Smear preparation: This is the primary stage, which involves the mixing of the inoculum
with a drop of sterile water and spreading it until a thin film is formed over the glass slide.
Fixation of smear: It is the second stage, which involves drying and heat fixing the thin
microbial layer formed on the glass slide.
Staining of the specimen: This is the final stage where the stain is applied onto the dried
smear, which imparts colour to the microscopic matter. This procedure is carried out prior
to microscopic examination and biochemical tests.
Fixation:
The stained cells seen in a microscope should resemble living cells as closely as possible.
Fixation is the process by which the internal and external structures of cells and
microorganisms are preserved and fixed in position. It inactivates enzymes that might
disrupt cell morphology and toughens cell structures so that they do not change during
staining and observation. Microorganism usually is killed and attached firmly to the
microscope slide during fixation. There are two fundamentally different types of fixation.
Heat fixation is routinely used to observe procaryotes. Typically, a film of cells (a smear)
is gently heated as a slide is passed through a flame. Heat fixation preserves overall
morphology but not structures within cells. Chemical fixation is used to protect fine
cellular substructure and the morphology of larger, more delicate microorganisms.
Chemical fixatives penetrate cells and react with cellular components, usually proteins and
lipids, to render them inactive, insoluble, and immobile.

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 Types of Stains
➢ Basic Stains: These stains are positively charged and bind to negatively charged
components of cells, such as nucleic acids and certain proteins. Examples include
methylene blue, crystal violet, and safranin.
1) Characteristics:
• Positively charged
• Bind to negatively charged components of cells
2) Examples:
• Methylene Blue: A basic dye commonly used in microbiology to stain cell nuclei
and certain bacteria.
• Crystal Violet: Used in Gram staining to stain bacterial cells.
• Safranin: Often used as a counterstain in Gram staining and as a general stain for
plant tissues
3) Applications:
• Staining cell nuclei
• Highlighting bacterial structures
• General staining in histology and cytology

➢ Acidic Stains: These stains are negatively charged and are repelled by negatively charged
cellular components. They are used to stain background material or structures that don't
readily take up basic stains. Examples include eosin and India ink.
1) Characteristics:
• Negatively charged
• Repelled by negatively charged cellular components
2) Examples:
• Eosin: A common acidic dye used in histology to stain cytoplasm, extracellular
matrix, and connective tissues.
• India Ink: Used for negative staining of bacterial capsules and in certain
microscopy applications.
3) Applications:
• Staining cytoplasm and extracellular matrix
• Negative staining techniques

Classification of stain
Stains can be classified based on various criteria, such as their chemical nature, staining
mechanism, target material, and specific application. Here’s an overview of the common
classifications:

1) Based on Chemical Properties

a) Acidic, Basic, and Neutral Stains
➢ Acidic Stains
• Properties: Contain negatively charged (anionic) chromophores.
• Examples: Eosin, Acid fuchsin.
• Application: Bind to positively charged components of cells, such as cytoplasm
and extracellular matrix proteins.
• Usage: Often used in combination with basic stains for contrast.
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➢ Basic Stains
• Properties: Contain positively charged (cationic) chromophores.
• Examples: Methylene blue, Crystal violet, Safranin.
• Application: Bind to negatively charged cell components, such as nucleic acids and
bacterial cell walls.
• Usage: Commonly used for staining bacteria and cell nuclei.
➢ Neutral Stains
• Properties: Combinations of acidic and basic dyes.
• Examples: Neutral red.
• Application: Can stain both acidic and basic cell components, often used for
staining cytoplasm and cell nuclei simultaneously.
2) Based on Biological Structures Targeted
a) Cytoplasmic Stains
• Examples: Eosin, Orange G.
• Application: Stains the cytoplasm of cells, often used in histology.
b) Nuclear Stains
• Examples: Hematoxylin, Methylene blue.
• Application: Stains cell nuclei, useful for identifying nuclear structures and chromatin.
c) Connective Tissue Stains
• Examples: Masson's trichrome, Van Gieson's stain.
• Application: Differentiates between muscle, collagen, and other connective tissues.
d) Carbohydrate Stains
• Examples: Periodic acid-Schiff (PAS) stain.
• Application: Highlights glycogen, mucins, and other polysaccharides.
e) Lipid Stains
• Examples: Oil Red O, Sudan IV.
• Application: Stains lipids in cells and tissues, often used in pathology to identify fat
accumulation.
3) Based on Application
a) Routine Stains
• Examples: Hematoxylin and Eosin (H&E) stain.
• Application: Widely used in medical diagnostics and research to provide general tissue
morphology.
b) Differential Stains
• Examples: Gram stain, Acid-fast stain.
• Application: Distinguishes between different types of microorganisms or cell types,
important for microbiology and pathology.
c) Special Stains
• Examples: Silver stain, Giemsa stain.
• Application: Used for specific structures or organisms, such as nerve fibers or blood
parasites.
4) Based on Mechanism of Staining
a) Physical Stains
• Examples: Sudan stains for lipids.
• Mechanism: Stains dissolve in and color the lipid structures without forming a
chemical bond.
b) Chemical Stains
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 • Examples: Hematoxylin (oxidized to form a complex with aluminum ions).
• Mechanism: Involves a chemical reaction between the stain and the tissue components.
5) Based on Solubility
a) Aqueous Stains
• Examples: Methylene blue, Eosin.
• Application: Used in water-based solutions, suitable for hydrated or aqueous
preparations.
b) Alcoholic Stains
• Examples: Crystal violet, Carbol fuchsin.
• Application: Used in alcohol-based solutions, often employed in procedures requiring
dehydration or fixation with alcohol.
6) Based on Fluorescence
1) Non-Fluorescent Stains
• Examples: Hematoxylin, Eosin.
• Application: Do not fluoresce, used in standard light microscopy.
2) Fluorescent Stains
• Examples: DAPI (4',6-diamidino-2-phenylindole), FITC (fluorescein isothiocyanate).
• Application: Emit light when excited by specific wavelengths, used in fluorescence
microscopy.
Theories of staining
Theories of staining provide explanations for how and why stains bind to biological
specimens. Several theories have been developed to describe the underlying mechanisms
of staining processes. Here are the major theories of staining:
1) Physical Theory
Description: This theory posits that staining occurs due to physical interactions between
the stain and the tissue or cell components.
➢ Mechanisms:
• Adsorption: Stains are adsorbed onto the surface of cells or tissues. The amount of
adsorption depends on the surface area and properties of the material being stained.
• Capillary Action: Capillary action allows stains to penetrate into tissues and cells,
especially in thin sections.
➢ Examples: Lipid stains like Sudan III and Oil Red O dissolve in lipids and stain them based
on their solubility.
2) Chemical Theory
Description: The chemical theory suggests that staining is the result of chemical reactions
between the stain and the tissue components.
➢ Mechanisms:
• Affinity: The dye has a specific affinity for certain cellular components due to chemical
interactions such as ionic bonds, covalent bonds, or hydrogen bonds.
• Complex Formation: Stains may form complexes with cellular components,
enhancing their visibility. For example, hematoxylin forms a complex with aluminum
ions, which then binds to nucleic acids in cell nuclei.
➢ Examples: Hematoxylin and Eosin (H&E) staining involves the formation of chemical
bonds between the dyes and tissue components. Hematoxylin binds to basophilic structures

15 | P a g e
 (nucleic acids) through complex formation, while eosin binds to eosinophilic structures
(proteins) through ionic interactions.
3) Electrostatic Theory (Ionic Theory)
Description: This theory is based on the principle that staining involves electrostatic
interactions between the charged components of the stain and the opposite charges in the
tissue or cell components.
➢ Mechanisms:
• Ionic Bonds: Basic dyes, which are positively charged, bind to negatively charged
structures like nucleic acids and acidic components of the cell. Acidic dyes, which are
negatively charged, bind to positively charged components like proteins and cytoplasm.
➢ Examples: Gram staining differentiates bacteria based on the differences in their cell wall
properties and their interactions with charged stains (crystal violet and safranin).
4) Histochemical Theory
This theory suggests that staining results from specific biochemical reactions between the
stain and certain chemical components within the tissues or cells.
➢ Mechanisms:
• Enzyme Reactions: Stains may react with specific enzymes within tissues, resulting in
a colored product that highlights specific structures.
• Chemical Specificity: The stain reacts with particular chemical groups present in the
tissue, such as polysaccharides or lipids.
➢ Examples: Periodic Acid-Schiff (PAS) stain reacts with polysaccharides, oxidizing
them to form aldehydes that subsequently react with Schiff reagent to produce a
magenta color. Feulgen reaction specifically stains DNA by hydrolyzing it to release
aldehyde groups that react with Schiff reagent.
5) Solubility Theory
Description: This theory posits that staining is based on the differential solubility of the
dye in the tissue components compared to the surrounding medium.
➢ Mechanisms:
• Lipid Solubility: Lipophilic stains dissolve in lipids and preferentially stain fatty
tissues and structures.
• Solvent Interaction: The solubility of the stain in the solvent and the tissue affects how
well the stain is taken up by different components.
➢ Examples: udan dyes stain lipids due to their high solubility in fat as compared to water.
6) Permeability Theory
Description: This theory focuses on the permeability of cell membranes and the differential
uptake of stains by various cellular components.
➢ Mechanisms:
• Selective Permeability: Differences in membrane permeability influence which stains
enter cells or organelles. Certain stains penetrate all cells, while others may selectively
stain based on membrane properties.
➢ Examples: Vital stains, such as trypan blue, selectively stain dead cells because their
membranes are permeable to the dye, whereas living cells exclude it.
7) Induced Affinity Theory

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 Description: This theory suggests that certain stains have an induced affinity for specific
cellular components due to interactions with other molecules or conditions that enhance
staining.
➢ Mechanisms:
• Co-staining: A mordant (a substance that enhances staining) is often used to increase
the binding affinity of the dye for the tissue. For instance, iodine in the Gram stain acts
as a mordant for crystal violet.
• Environmental Conditions: pH, temperature, and other environmental factors can
influence the affinity of the stain for specific cellular components.
➢ Examples: Gram staining involves the use of iodine as a mordant to form a complex with
crystal violet, which enhances the staining of Gram-positive bacteria.
Mechanism of Gram Staining
Purpose: Differentiates bacteria into Gram-positive and Gram-negative based on cell wall
composition.
1) Primary Stain (Crystal Violet): Stains all bacterial cells purple.
2) Mordant (Iodine): Forms a crystal violet-iodine complex within the cells.
3) Decolorization (Alcohol/Acetone):
• Gram-positive cells retain the crystal violet-iodine complex due to their thick
peptidoglycan layer, remaining purple.
• Gram-negative cells lose the crystal violet-iodine complex because the alcohol
dissolves the outer membrane and thin peptidoglycan layer, becoming colorless.
4) Counterstain (Safranin):
• Stains the decolorized Gram-negative cells pink.
• Gram-positive cells remain purple as the crystal violet-iodine complex masks the pink
stain.

Mechanism of Acid-Fast Staining (Ziehl-Neelsen Stain)

Purpose: Identifies acid-fast bacteria (e.g., Mycobacterium species) with waxy cell walls
containing mycolic acids.

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1) Primary Stain (Carbol Fuchsin): Applied with heat (steam) to penetrate the waxy cell
wall, staining all cells red.
2) Decolorization (Acid Alcohol):
• Acid-fast bacteria retain the carbol fuchsin due to their waxy, lipid-rich cell walls,
remaining red.
• Non-acid-fast bacteria lose the carbol fuchsin and become colorless.
3) Counterstain (Methylene Blue):
• Stains non-acid-fast cells blue.
• Acid-fast cells remain red.

Mechanism of Negative Staining

Purpose: Visualizes the shape and size of bacteria, especially for observing capsules,
without penetrating the cells.
1) Stain (Nigrosin or India Ink): Mix the bacterial sample with a drop of stain on a slide.
Spread the mixture to create a thin film. Stain does not penetrate the cells but stains the
background, leaving cells colorless against a dark background.
2) Drying: Air dry the slide without heat fixing to prevent distortion of the cells.

Mechanism of Capsule Staining

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 Purpose: Visualizes bacterial capsules, which are usually not stained by basic stains.
1) Negative Stain (Congo Red or India Ink): Stains the background, leaving capsules clear.
2) Positive Stain (Maneval's Stain): Stains the bacterial cell, but not the capsule.
3) Result: Capsules appear as clear halos around the stained cells against a dark or colored
background.

Mechanism of Flagella Staining

Purpose: Visualizes bacterial flagella, which are too thin to be seen with standard stains.
1) Mordant (Tannic Acid or Potassium Alum): Coats the flagella to increase their diameter.
2) Stain (Carbol Fuchsin or Silver Nitrate): Stains the thickened flagella.
3) Result: Flagella are visible as stained structures extending from the cells.

Mechanism of Endospore Staining (Schaeffer-Fulton Method)

Purpose: Visualizes bacterial endospores, which are resistant to ordinary staining methods.
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1) Primary Stain (Malachite Green): Applied with heat (steam) to penetrate the tough spore
coat, staining endospores green.
2) Decolorization (Water): Removes the stain from vegetative cells but not from endospores.
3) Counterstain (Safranin): Stains the decolorized vegetative cells pink. Endospores remain
green.

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 Module-3
Microbial Growth and Nutrition
Microbial Growth
Microbial growth refers to an increase in the number of cells, not in the size of individual
cells. This process involves cell division, usually by binary fission, where one cell splits
into two identical daughter cells.
➢ Phases of Microbial Growth

1) Lag Phase: Cells adapt to a new environment. Little to no cell division. Cells are
metabolically active, synthesizing enzymes and molecules necessary for growth.

2) Log (Exponential) Phase: Rapid cell division and exponential growth. Cells are most
active metabolically. Generation time (time required for a cell to divide) is constant.

3) Stationary Phase: Growth rate slows down. Nutrient depletion and accumulation of waste
products. Number of new cells equals the number of dying cells.

4) Death (Decline) Phase: Number of dying cells exceeds the number of new cells formed.
Nutrients are depleted, and waste products become toxic.

Nutritional Requirements of Microorganisms

Microorganisms require various nutrients for energy production and cellular biosynthesis.
These nutrients can be classified into several categories:

1) Macronutrients: Required in large quantities. Include carbon, nitrogen, phosphorus,

sulfur, potassium, magnesium, calcium, and iron.

2) Micronutrients (Trace Elements): Required in trace amounts. Include elements like zinc,
copper, cobalt, manganese, and molybdenum. Often function as cofactors in enzymatic
reactions.

3) Growth Factors: Organic compounds that microorganisms cannot synthesize but need for
growth. Include vitamins, amino acids, purines, and pyrimidines.

Common nutrient requirements

Microorganisms, like all living organisms, have specific nutrient requirements to support
their growth, metabolism, and reproduction. These requirements can be categorized into
macronutrients and micronutrients. Here’s a detailed look at the common nutrient
requirements:
➢ Macronutrients

1) Carbon (C): Primary building block of cellular material, energy source.

• Sources: Organic compounds (e.g., glucose for heterotrophs), carbon dioxide (CO2 for
autotrophs).
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2) Nitrogen (N): Essential for amino acids, proteins, nucleic acids, and other cell constituents.
• Sources: Ammonia (NH3), nitrate (NO3-), nitrogen gas (N2), and organic nitrogen
compounds.

3) Phosphorus (P): Critical for nucleic acids, ATP, and membrane phospholipids.
• Sources: Inorganic phosphate (PO4^3-), organic phosphates.

4) Sulfur (S): Required for the synthesis of certain amino acids (e.g., cysteine and methionine)
and vitamins.
• Sources: Sulfate (SO4^2-), hydrogen sulfide (H2S), sulfur-containing amino acids.

5) Potassium (K): Enzyme activation, maintaining osmotic balance.

• Sources: Potassium salts (e.g., KCl).

6) Magnesium (Mg): Enzyme cofactor, stabilizes ribosomes, membranes, and nucleic acids.
• Sources: Magnesium salts (e.g., MgSO4).

7) Calcium (Ca): Stabilizes cell walls, endospores formation in some bacteria.

• Sources: Calcium salts (e.g., CaCl2).

8) Iron (Fe): Component of cytochromes and enzymes involved in electron transport.

• Sources: Ferrous (Fe2+) and ferric (Fe3+) salts, siderophores to scavenge iron.
➢ Micronutrients (Trace Elements)
These are required in much smaller amounts but are essential for various cellular processes,
particularly as enzyme cofactors.

1) Manganese (Mn): Enzyme cofactor, protection against oxidative damage.

• Sources: Manganese salts.

2) Zinc (Zn): Component of many enzymes, including DNA-binding proteins.

• Sources: Zinc salts.

3) Cobalt (Co): Component of vitamin B12.

• Sources: Cobalt salts.

4) Molybdenum (Mo): Component of enzymes involved in nitrogen fixation and reduction.

• Sources: Molybdenum salts.

5) Nickel (Ni): Component of some hydrogenases and enzymes involved in methane

production.
• Sources: Nickel salts.

6) Copper (Cu): Component of cytochrome c oxidase and other enzymes.

• Sources: Copper salts.
➢ Growth Factors
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 These are organic compounds that some microorganisms cannot synthesize and must obtain
from the environment:

1) Vitamins: Coenzymes and precursors for coenzymes (e.g., B vitamins).

• Sources: Environmentally available vitamins or provided in growth media.

2) Amino Acids: Building blocks of proteins.

• Sources: External amino acids if the microorganism cannot synthesize them.

3) Purines and Pyrimidines: Building blocks of nucleic acids (DNA and RNA).
• Sources: Exogenous purines and pyrimidines if needed.
➢ Nutritional Types
Microorganisms can be classified into different nutritional types based on their sources of
carbon, energy, and electrons. Here's an overview of the primary nutritional types, along
with their definitions and examples:

1) Based on Carbon Source

Autotrophs: Organisms that utilize inorganic carbon (usually carbon dioxide, CO2) as
their primary carbon source.
❖ Examples:
• Photoautotrophs: Use light as an energy source. Example: Cyanobacteria.
• Chemoautotrophs: Use inorganic compounds as an energy source. Example:
Nitrifying bacteria (e.g., Nitrosomonas).
Heterotrophs: Organisms that require organic carbon (e.g., glucose) for growth.
❖ Examples:
• Photoheterotrophs: Use light as an energy source but require organic compounds for
carbon. Example: Purple non-sulfur bacteria (e.g., Rhodospirillum).
• Chemoheterotrophs: Obtain both energy and carbon from organic compounds.
Example: Most bacteria, fungi, protozoa, and animals.

2) Based on Energy Source

Phototrophs: Organisms that use light as their primary energy source.
❖ Examples:
•Photoautotrophs: Use light for energy and CO2 for carbon. Example: Algae,
cyanobacteria.
• Photoheterotrophs: Use light for energy and organic compounds for carbon. Example:
Purple non-sulfur bacteria.
Chemotrophs: Organisms that obtain energy from chemical compounds.
❖ Examples:
• Chemoautotrophs: Use inorganic compounds for both energy and CO2 for carbon.
Example: Sulfur-oxidizing bacteria (e.g., Thiobacillus).
• Chemoheterotrophs: Use organic compounds for both energy and carbon. Example:
Most pathogenic bacteria (e.g., Escherichia coli).

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3) Based on Electron Source
Lithotrophs: Organisms that use inorganic molecules as a source of electrons.
❖ Examples:
• Chemoautotrophic Lithotrophs: Use inorganic compounds for both energy and
electrons and CO2 for carbon. Example: Nitrosomonas (nitrifying bacteria).
• Photoautotrophic Lithotrophs: Use light for energy, inorganic molecules for
electrons, and CO2 for carbon. Example: Cyanobacteria.
Organotrophs: Organisms that use organic molecules as a source of electrons.
❖ Examples:
• Chemoheterotrophic Organotrophs: Use organic compounds for energy, carbon, and
electrons. Example: Most fungi, animals, and many bacteria (e.g., E. coli).
• Photoheterotrophic Organotrophs: Use light for energy and organic molecules for
carbon and electrons. Example: Some purple non-sulfur bacteria.

Culture Media
Culture media are crucial for the cultivation, isolation, and identification of microorganisms
in a laboratory setting. Different types of culture media are designed to meet the specific
growth requirements of various microorganisms. Here's a detailed overview of the main
types of culture media:

1) Basic (Simple) Media: Basic or simple media provide the essential nutrients required for
the general growth of microorganisms. They are often used for routine cultivation of
microbes.
Components:
• Peptones
• Meat extract or yeast extract
• Water
• Agar (for solid media)
Examples:
• Nutrient Agar (NA): Commonly used for growing a wide variety of non-fastidious
organisms.
• Nutrient Broth: The liquid form of nutrient agar without agar, used for the cultivation of
bacteria in a liquid environment.
2) Enriched Media: Enriched media contain additional nutrients to support the growth of
fastidious organisms that require specific nutrients not present in basic media.
Components:
• Basic media components
• Blood, serum, or growth factors
Examples:
• Blood Agar: Enriched with whole blood (usually sheep or horse blood), used to grow
fastidious bacteria and to detect hemolytic activity.

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• Chocolate Agar: Contains lysed blood, providing additional nutrients required by certain
fastidious bacteria such as Haemophilus influenzae and Neisseria species.
3) Selective Media: Selective media favor the growth of particular microorganisms while
inhibiting the growth of others. They are used for isolating specific types of bacteria from
mixed cultures.
Components:
• Basic media components
• Selective agents (antibiotics, dyes, salts)
Examples:
• MacConkey Agar: Selective for Gram-negative bacteria due to the presence of bile salts
and crystal violet which inhibit Gram-positive bacteria.
• Mannitol Salt Agar (MSA): Selective for staphylococci due to high salt concentration
(7.5% NaCl) that inhibits most other bacteria.
4) Differential Media: Differential media contain indicators that allow differentiation
between different types of microorganisms based on their biochemical properties.
Components:
• Basic media components
• Indicators (pH indicators, dyes)
• Specific substrates (carbohydrates, amino acids)
Examples:
• MacConkey Agar: Differentiates lactose fermenters (which turn the medium pink) from
non-lactose fermenters (which leave it colourless).
• Eosin Methylene Blue (EMB) Agar: Differentiates based on lactose fermentation, with
strong fermenters producing dark colonies with a metallic green sheen (e.g., E. coli).
5) Selective and Differential Media: These media combine the properties of both selective
and differential media, facilitating the isolation and differentiation of specific organisms
simultaneously.
Examples:
• MacConkey Agar: Both selective (for Gram-negative bacteria) and differential (lactose
fermentation).
• Mannitol Salt Agar (MSA): Selective for staphylococci and differential for mannitol
fermentation (mannitol fermenters turn the medium yellow).
6) Transport Media: Transport media are used to preserve specimens during transportation
to the laboratory without allowing the overgrowth of contaminating organisms or loss of
viability.
Components:
• Buffers
• Salts
• Reducing agents (to maintain anaerobic conditions, if necessary)
• No nutrients to prevent microbial multiplication

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 Examples:
• Stuart Transport Medium: Maintains the viability of organisms like Neisseria
gonorrhoeae.
• Cary-Blair Transport Medium: Used for transporting stool specimens, particularly for
enteric pathogens.
7) Anaerobic Media: Anaerobic media are designed to support the growth of anaerobic
microorganisms by providing an oxygen-free environment.
Components:
• Reducing agents (e.g., thioglycollate or cysteine)
• Nutrient-rich components to support growth
• Often used in conjunction with anaerobic jars or chambers to create an anaerobic
environment
Examples:
• Thioglycollate Broth: Contains sodium thioglycollate which reduces oxygen, allowing the
growth of anaerobes.
• Anaerobic Blood Agar: Enriched medium that supports anaerobic bacterial growth when
incubated in anaerobic conditions.
8) Specialized Media: Specialized media are formulated for the cultivation of specific types
of microorganisms or for the detection of specific microbial properties.
Examples:
• Lowenstein-Jensen Medium: Used for the cultivation of Mycobacterium tuberculosis.
• Sabouraud Dextrose Agar (SDA): Specialized for the cultivation of fungi, with a low pH
that inhibits most bacteria.
• CLED (Cystine-Lactose-Electrolyte-Deficient) Agar: Used for culturing urinary
pathogens and differentiating them based on lactose fermentation without electrolyte
inhibition of swarming bacteria like Proteus species.
Isolation of Pure Cultures
The goal of isolating pure cultures is to obtain a single type of microorganism free from
contaminants. This is crucial for studying its characteristics, behavior, and potential
applications.

1) Streak Plate Method:

• Procedure: A sterile inoculating loop is dipped into a sample containing multiple
microorganisms and streaked across the surface of an agar plate in a pattern that thins
out the sample over successive streaks.
• Result: Individual cells are separated on the agar surface, and after incubation, they
form isolated colonies.

2) Pour Plate Method:

• Procedure: A diluted microbial sample is mixed with molten agar and poured into a
petri dish. Once the agar solidifies, the plate is incubated.
• Result: Colonies grow both on the surface and within the agar, allowing for isolation
of pure colonies from different depths.

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3) Spread Plate Method:
• Procedure: A diluted microbial sample is spread evenly across the surface of an agar
plate using a sterile spreading tool.
• Result: Colonies grow on the surface, and isolated colonies can be selected for pure
culture.

4) Single Cell Isolation (Micromanipulation):

• Procedure: Individual cells are physically separated using a micromanipulator under a
microscope.
• Result: Single cells are transferred to a sterile medium to grow into pure cultures.
Cultivation of Pure Cultures
Cultivation allows the growth and multiplication of microorganisms under controlled
conditions, providing the necessary environment and nutrients.

1) Batch Culture:
• Description: Microorganisms are grown in a closed system with a fixed volume of
nutrient medium.
• Example: Growth in flasks or test tubes.

2) Continuous Culture:
• Description: Fresh medium is continuously added to the culture while an equal volume
of spent medium is removed, maintaining the culture in a steady state.
• Example: Chemostat, which keeps the microbial population in the exponential growth
phase.

3) Solid Media Cultures:

• Description: Microorganisms are grown on the surface of solidified agar media in petri
dishes.
• Example: Streak plates, slant cultures.

4) Liquid Media Cultures:

• Description: Microorganisms are grown in liquid broth, which allows for easy
sampling and measurement of growth.
• Example: Shake flasks, fermentation broths.
Maintenance of Pure Cultures
1) Regular Subculturing:
• Procedure: Microorganisms are periodically transferred (subcultured) to fresh media to
maintain active growth and viability.
• Frequency: Typically done every few weeks to months, depending on the organism and
medium.
• Drawbacks: Risk of contamination, labor-intensive, and potential for genetic drift over
time.
2) Refrigeration:
• Procedure: Cultures are stored at 4°C (refrigerator temperature) to slow down metabolic
activity and growth.

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• Media: Solid media (agar slants) or liquid media (broths) can be used.
• Drawbacks: Limited storage time (weeks to months) and some microorganisms may not
survive well at this temperature.
3) Mineral Oil Overlay:
• Procedure: Agar slants are covered with sterile mineral oil to prevent desiccation and limit
oxygen exposure.
• Temperature: Typically stored at room temperature or in the refrigerator.
• Advantages: Can extend the viability of cultures for several months to a year.
• Drawbacks: Not suitable for all microorganisms and may complicate subsequent culturing.
Preservation of Pure Cultures
1) Freezing:
• Procedure: Cultures are mixed with a cryoprotectant (e.g., glycerol, dimethyl sulfoxide)
and stored at ultra-low temperatures.
• Storage Temperatures:
a) Standard Freezers (-20°C): Short-term storage.
b) Ultra-Low Freezers (-80°C): Long-term storage.
• Advantages: Maintains viability and genetic stability for several years.
• Drawbacks: Requires specialized equipment and cryoprotectants.
2) Lyophilization (Freeze-Drying):
• Procedure: Cultures are frozen and then dehydrated under vacuum to remove moisture.
• Storage: The resulting dried material can be sealed in ampoules or vials and stored at room
temperature or in a refrigerator.
• Advantages: Very long-term storage (decades), easy transport, and reconstitution.
• Drawbacks: Initial setup and equipment are expensive, and some microorganisms may not
survive the process.
3) Cryopreservation:
• Procedure: Cells are suspended in a cryoprotective solution and stored in liquid nitrogen
(-196°C) or in ultra-low freezers.
• Advantages: Provides excellent long-term preservation (indefinitely), maintaining cell
viability and genetic stability.
• Drawbacks: High initial cost and the need for continuous liquid nitrogen supply or ultra-
low freezers.
4) Desiccation:
• Procedure: Cultures are dried on sterile filter papers or silica gel and stored in sealed
containers.
• Advantages: Simple and cost-effective, suitable for spore-forming bacteria and fungi.
• Drawbacks: Limited to certain types of microorganisms and storage times.
5) Agar Slant Storage:
• Procedure: Cultures are grown on agar slants, sealed with parafilm or rubber stoppers, and
stored in the refrigerator.
• Advantages: Simple and low-cost.
• Drawbacks: Short to medium-term storage, requires periodic subculturing.
Best Practices for Maintenance and Preservation
1) Aseptic Technique: Always use sterile techniques to avoid contamination during handling,
subculturing, and preservation.

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2) Labeling: Clearly label all cultures with relevant information, including species/strain
name, date of preservation, and storage conditions.
3) Documentation: Keep detailed records of the preservation method, medium used, and any
observations regarding the culture's viability and characteristics.
4) Regular Checks: Periodically check preserved cultures for viability and contamination.
This can involve reviving a small sample and ensuring it grows as expected.
5) Genetic Stability: Minimize the number of subculturing steps to reduce the risk of genetic
drift. Preserve multiple aliquots to avoid repeated thawing and freezing.
Growth Curve
A microbial growth curve represents the growth of a microbial population over time,
typically plotted as the logarithm of the number of cells against time. In a closed system
(batch culture), the growth curve typically consists of four distinct phases:

1) Lag Phase:
• Description: A period of adaptation where microorganisms adjust to the new
environment.
• Activity: Cells are metabolically active but not dividing. They synthesize enzymes,
nucleic acids, and other molecules necessary for growth.
• Duration: Varies depending on the previous growth conditions and the nature of the
medium.

2) Log (Exponential) Phase:

• Description: A period of rapid
and constant cell division.
• Activity: Cells are actively
dividing and the population
size doubles at a constant rate.
• Growth Rate: Characterized
by the maximum growth rate
(μmax) and is used to calculate
generation time.
• Importance: Ideal for
studying microbial physiology
and conducting experiments.

3) Stationary Phase:
• Description: The growth rate slows as nutrients deplete and waste products
accumulate.
• Activity: The rate of cell division equals the rate of cell death. The total number of
viable cells remains constant.
• Causes: Nutrient limitation, oxygen depletion, accumulation of toxic byproducts, and
changes in pH.

4) Death (Decline) Phase:

• Description: The number of viable cells decreases exponentially.

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 • Activity: Cell death exceeds cell division due to severe nutrient depletion and toxic
environment.
• Importance: Relevant for studying cell death and survival mechanisms.
Generation Time
Generation time (doubling time) is the time it takes for a microbial population to double
in number during the exponential phase. It is a measure of the growth rate and varies among
different microorganisms and environmental conditions.

1) Calculation:
g=n/t
Where:
o g = generation time
o t = time period of exponential growth
o n = number of generations

2) Determining Number of Generations (n):

n=(logNt−logN0)/ log2
Where:
o Nt = number of cells at time ttt
o N= initial number of cells

3) Growth Rate Constant (k):

k=1/g
Alternatively, k can be expressed as:
k=(logNt−logN0)/ 0.301×t

Batch Culture
Batch culture is a closed-system culture method where microorganisms are grown in a
fixed volume of nutrient medium without additional nutrient supply once the culture is
initiated.

1) Characteristics:
• Nutrient Supply: Finite and not replenished during the growth period.
• Phases: Exhibits the typical growth curve phases (lag, log, stationary, death).
• Applications: Used in laboratory experiments, fermentation processes, and studying
microbial growth kinetics.

2) Advantages:
• Simple to set up and manage.
• Useful for producing microbial metabolites, enzymes, and studying growth dynamics.

3) Disadvantages:
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• Limited by nutrient depletion and waste accumulation.
• Cannot maintain cells in the exponential phase for extended periods.
➢ Synchronous and Continuous Culture
Synchronous Culture
Synchronous culture is a technique where all the cells in a culture are at the same stage of
the cell cycle. This synchronization allows for detailed studies of cellular processes, such
as DNA replication and cell division, at specific times during the cell cycle.

1) Methods to Achieve Synchronization:

• Temperature Shifts: Alternating between permissive and restrictive temperatures to
synchronize cells.
• Nutrient Deprivation and Resupply: Starving cells of a crucial nutrient and then
reintroducing it to synchronize growth.
• Chemical Inhibitors: Using chemicals to block cell cycle progression and then removing
the inhibitor to allow synchronized growth.
• Density Gradient Centrifugation: Separating cells based on size and stage of the cell
cycle using centrifugation.

2) Applications:
• Studying cell cycle events.
• Investigating the effects of drugs on specific stages of the cell cycle.
• Understanding microbial physiology under controlled conditions.
Continuous Culture
Continuous culture maintains microbial cells in a steady state of growth over extended
periods by continuously supplying fresh nutrients and removing waste products. This is
achieved in a bioreactor system.

1) Types of Continuous Culture Systems:

• Chemostat: Maintains a constant culture volume by continuously adding fresh medium at
the same rate as the culture is removed. The growth rate is controlled by the dilution rate
(rate at which fresh medium is added).
• Turbidostat: Maintains a constant cell density by adjusting the dilution rate based on the
turbidity (optical density) of the culture.

2) Advantages:
• Steady-state conditions allow for precise control of growth rate and nutrient concentration.
• Ideal for studying microbial kinetics and metabolic processes.
• Useful in industrial applications for producing microbial metabolites, enzymes, and other
products.

3) Disadvantages: Requires careful monitoring and control. Risk of contamination over long
periods.

Measurement of Microbial Growth

1) Direct Methods:
a) Microscopic Counts:
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 o Procedure: Counting cells under a microscope using a counting chamber (e.g.,
hemocytometer).
b) Viable Plate Counts:
o Procedure: Diluting the sample and spreading it on an agar plate to count colony-
forming units (CFUs).
c) Most Probable Number (MPN):
o Procedure: Estimating cell numbers based on statistical probability from multiple
dilutions.

2) Indirect Methods:
a) Turbidimetric Measurements:
o Procedure: Measuring the turbidity (cloudiness) of a culture using a
spectrophotometer.
b) Dry Weight:
o Procedure: Drying and weighing the biomass of a culture.
c) Metabolic Activity:
o Procedure: Measuring the rate of a specific metabolic process (e.g., oxygen
consumption, CO2 production).

Factors Influencing Microbial Growth

1) Physical Factors:
a) Temperature:
o Psychrophiles: Grow best at low temperatures (0-15°C).
o Mesophiles: Grow best at moderate temperatures (20-45°C).
o Thermophiles: Grow best at high temperatures (45-80°C).
o Hyperthermophiles: Grow at extremely high temperatures (>80°C).

b) pH:
o Acidophiles: Prefer acidic conditions (pH < 6).
o Neutrophiles: Prefer neutral conditions (pH 6-8).
o Alkaliphiles: Prefer alkaline conditions (pH > 8).

c) Oxygen:
o Obligate Aerobes: Require oxygen for growth.
o Obligate Anaerobes: Cannot tolerate oxygen.
o Facultative Anaerobes: Can grow with or without oxygen.
o Aerotolerant Anaerobes: Do not require oxygen but are not harmed by its
presence.

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 o Microaerophiles: Require low levels of oxygen.

d) Osmotic Pressure:
o Halophiles: Thrive in high salt concentrations.
o Osmophiles: Prefer environments with high sugar concentrations.

2) Chemical Factors:
a) Nutrients:
o Carbon: Essential for all organic molecules.
o Nitrogen: Needed for amino acids and nucleic acids.
o Phosphorus: Important for nucleic acids and ATP.
o Sulfur: Required for some amino acids and vitamins.
o Trace Elements: Metals like iron, manganese, and zinc, which act as enzyme
cofactors.
b) Growth Factors:
o Vitamins: Organic compounds necessary in small amounts.
o Amino Acids: Required for protein synthesis.
o Purines and Pyrimidines: Necessary for nucleic acid synthesis.

Classification of Microbes Based on Oxygen Requirement

1) Obligate Aerobes:
•Description: Require oxygen for growth and survival. They use oxygen as the terminal
electron acceptor in aerobic respiration.
• Example: Mycobacterium tuberculosis, Pseudomonas aeruginosa.
• Adaptations: Possess enzymes like superoxide dismutase (SOD) and catalase that
detoxify reactive oxygen species (ROS).
2) Obligate Anaerobes:
• Description: Cannot tolerate oxygen and may even be killed by it. They typically rely
on anaerobic respiration or fermentation for energy production.
• Example: Clostridium botulinum, Bacteroides fragilis.
• Adaptations: Lack enzymes like superoxide dismutase and catalase, making them
vulnerable to oxidative damage.
3) Facultative Anaerobes:
• Description: Can grow in the presence or absence of oxygen. They use aerobic
respiration when oxygen is available but can switch to anaerobic respiration or
fermentation when it is not.
• Example: Escherichia coli, Saccharomyces cerevisiae.
• Adaptations: Possess both aerobic and anaerobic metabolic pathways, and enzymes to
detoxify ROS.
4) Aerotolerant Anaerobes:
• Description: Do not require oxygen for growth but can tolerate its presence. They
typically rely on fermentation for energy production.

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 • Example: Lactobacillus spp., Streptococcus pyogenes.
• Adaptations: Possess enzymes like superoxide dismutase that protect against oxidative
damage but lack a functional electron transport chain for aerobic respiration.
5) Microaerophiles:
• Description: Require low levels of oxygen (2-10%) for growth but are inhibited by
higher concentrations. They perform aerobic respiration.
• Example: Helicobacter pylori, Campylobacter jejuni.
• Adaptations: Have a lower tolerance for ROS and limited or less efficient detoxifying
enzymes compared to obligate aerobes.
Microbial Metabolism
Microbial metabolism encompasses the chemical reactions that occur within
microorganisms to maintain life, providing energy and building blocks for growth and
reproduction. It includes catabolic pathways, which break down molecules to release
energy, and anabolic pathways, which use energy to synthesize complex molecules. Some
pathways function in both catabolism and anabolism, known as amphibolic pathways.
Here’s an overview of these concepts:

Metabolic Pathways
1) Catabolic Pathways:
• Purpose: Degrade complex molecules into simpler ones, releasing energy stored in
chemical bonds.
• Examples:
a) Glycolysis: The breakdown of glucose to pyruvate, producing ATP and NADH.
b) Krebs Cycle (Citric Acid Cycle): Oxidation of acetyl-CoA to CO2, generating ATP,
NADH, and FADH2.
c) Electron Transport Chain (ETC): Transfer of electrons from NADH and FADH2 to
oxygen (in aerobes), producing ATP through oxidative phosphorylation.
d) Fermentation: Anaerobic process that regenerates NAD+ by converting pyruvate to
lactic acid, ethanol, or other byproducts.
2) Anabolic Pathways:
• Purpose: Synthesize complex molecules from simpler ones, requiring energy input.
• Examples:
a) Photosynthesis: Conversion of light energy into chemical energy stored in glucose.
b) Calvin Cycle: Synthesis of glucose from CO2 and water using ATP and NADPH
generated in the light reactions of photosynthesis.
c) Amino Acid Biosynthesis: Formation of amino acids from intermediates of glycolysis,
the Krebs cycle, or the pentose phosphate pathway.
d) Nucleotide Biosynthesis: Synthesis of nucleotides from amino acids, ribose-5-
phosphate, CO2, and ammonia.
Amphibolic Pathways
Amphibolic pathways serve dual roles in both catabolism and anabolism, facilitating the
integration and regulation of metabolic processes.
1) Krebs Cycle (Citric Acid Cycle):
• Catabolic Role: Oxidizes acetyl-CoA to CO2, producing ATP, NADH, and FADH2.

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 • Anabolic Role: Provides precursors for the synthesis of amino acids, nucleotides, and
other biosynthetic pathways.
• Intermediates: Compounds like oxaloacetate and α-ketoglutarate are precursors for
amino acid synthesis.
2) Glycolysis and Gluconeogenesis:
• Glycolysis: Catabolic pathway breaking down glucose to pyruvate, generating ATP.
• Gluconeogenesis: Anabolic pathway synthesizing glucose from non-carbohydrate
precursors like pyruvate and lactate.
• Shared Intermediates: Compounds like glucose-6-phosphate and fructose-6-
phosphate are involved in both pathways.
Biosynthetic Pathways
Biosynthetic pathways involve the construction of complex molecules needed for cell
growth and function.
1) Amino Acid Biosynthesis:
• Pathways: Synthesis of amino acids from intermediates of central metabolic pathways.
• Examples: Glutamate and glutamine synthesis from α-ketoglutarate; serine synthesis from
3-phosphoglycerate.
2) Nucleotide Biosynthesis:
• Purine and Pyrimidine Synthesis: Construction of nucleotides from amino acids, ribose-
5-phosphate, CO2, and ammonia.
• Examples: Synthesis of adenine and guanine (purines), cytosine, thymine, and uracil
(pyrimidines).
3) Lipid Biosynthesis:
• Fatty Acid Synthesis: Formation of fatty acids from acetyl-CoA and malonyl-CoA.
• Phospholipid Synthesis: Construction of phospholipids from glycerol-3-phosphate and
fatty acids.
• Steroid Biosynthesis: Synthesis of steroids like cholesterol from acetyl-CoA.

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 Module-4
Bacterial Recommendation
➢ Bacterial gene transfer
Transformation
Transformation is a process of horizontal gene transfer where bacteria take up extracellular
DNA from their environment and integrate it into their own genome. This process can
significantly contribute to genetic diversity and adaptability in bacterial populations.
Here's a detailed overview of the transformation process:
❖ Features of Transformation
1) Natural Competence: Some bacteria have a natural ability to take up DNA from their
environment, a state known as competence. Competence can be a transient state, influenced
by environmental factors such as nutrient availability, cell density, and stress conditions.
Competence can involve the expression of specific genes that encode for proteins
facilitating DNA uptake and integration.
2) DNA Uptake: Competent bacteria can bind extracellular DNA through surface receptors.
The DNA is then transported across the cell membrane(s). In Gram-positive bacteria, DNA
crosses the single cell membrane, while in Gram-negative bacteria, it must cross both the
outer and inner membranes.
3) Integration: Once inside the cell, the foreign DNA can be degraded into smaller fragments
or recombined into the host genome through homologous recombination. Homologous
recombination involves the exchange of genetic material between the foreign DNA and the
host chromosome at regions of sequence similarity.
4) Expression: If the foreign DNA is successfully integrated, the new genes can be expressed,
potentially conferring new traits such as antibiotic resistance, metabolic capabilities, or
virulence factors.

Mechanisms of Transformation
1) Natural Transformation: Observed in certain bacterial species such as Streptococcus
pneumoniae, Neisseria gonorrhoeae, and Bacillus subtilis. Involves the natural uptake of
naked DNA from the environment.
2) Artificial Transformation: Techniques developed to induce competence in bacteria that
are not naturally competent. Common methods include chemical induction using calcium
chloride and heat shock, or electroporation, where an electric field increases cell membrane
permeability to DNA.
➢ Stages of Transformation
1) Development of Competence: Induction of competence-specific genes in response to
environmental cues. Production of DNA-binding proteins and nucleases.
2) Binding of DNA: Extracellular DNA binds to specific receptors on the bacterial surface.
3) DNA Uptake: DNA is transported into the cell, often in single-stranded form, while the
complementary strand is degraded. Transport mechanisms involve membrane-spanning
complexes and energy-dependent processes.
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4) Integration and Recombination: Internalized DNA aligns with homologous regions of
the bacterial chromosome. Enzymes such as RecA facilitate homologous recombination,
allowing the new DNA to be incorporated into the host genome.

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 Transduction
Transduction is a method of horizontal gene transfer in bacteria mediated by bacteriophages
(viruses that infect bacteria). It plays a crucial role in bacterial evolution, genetic diversity,
and the spread of genes, including those responsible for antibiotic resistance and virulence.
There are two main types of transduction: generalized and specialized.

➢ Generalized Transduction
Generalized transduction can transfer any gene from the donor bacterium to the recipient
bacterium. It occurs during the lytic cycle of a bacteriophage.
1) Infection: A bacteriophage infects a bacterial cell (the donor).
2) Lytic Cycle: The phage commandeers the bacterial machinery to replicate its own DNA
and produce new phage particles.
3) DNA Fragmentation: The host bacterial chromosome is fragmented into smaller pieces.
4) Packaging Error: Occasionally, bacterial DNA fragments are mistakenly packaged into a
new phage particle instead of phage DNA.
5) Release: The newly formed phages, some containing bacterial DNA, are released when the
host cell lyses.
6) Transduction: A phage carrying bacterial DNA infects another bacterial cell (the
recipient), injecting the bacterial DNA.
7) Integration: The introduced DNA can recombine with the recipient's chromosome through
homologous recombination.

➢ Specialized Transduction
Specialized transduction involves the transfer of specific bacterial genes and occurs
during the lysogenic cycle of a bacteriophage.
1) Lysogenic Cycle: A temperate phage infects a bacterial cell and integrates its DNA into the
host genome, becoming a prophage.
2) Prophage Induction: When the prophage is excised from the bacterial chromosome to
enter the lytic cycle, it may mistakenly excise adjacent bacterial genes along with its own
DNA.
3) Packaging: These bacterial genes are packaged into new phage particles along with phage
DNA.
4) Infection: The phage carrying both viral and bacterial DNA infects another bacterial cell.
5) Integration: The bacterial genes from the phage can recombine with the recipient's
genome.
➢ Key Differences Between Generalized and Specialized Transduction
1) Generalized Transduction:
• Can transfer any part of the bacterial genome.
• Occurs due to accidental packaging of bacterial DNA during the lytic cycle.

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2) Specialized Transduction:
• Transfers specific genes located near the prophage integration site.
• Occurs due to improper excision of the prophage during induction from the lysogenic
cycle.

Conjugation
Conjugation is a method of horizontal gene transfer in bacteria that involves the direct
transfer of genetic material from a donor bacterium to a recipient bacterium. This process
requires physical contact between the two bacterial cells and is mediated by a specialized
structure called a sex pilus. Conjugation is a significant mechanism for the spread of
antibiotic resistance genes among bacterial populations and plays a crucial role in bacterial
evolution and adaptation.
➢ Features of Conjugation
1) Donor Cell: The donor bacterium contains a conjugative plasmid, which carries the genes
necessary for conjugation, often referred to as the F (fertility) plasmid.
2) Conjugative Pilus Formation:
• The donor bacterium synthesizes a proteinaceous appendage called the sex pilus,
encoded by the F plasmid.
• The sex pilus extends from the donor cell and makes contact with a recipient bacterium.
3) DNA Transfer:
• The F plasmid contains genes that encode for the formation of a mating bridge between
the donor and recipient cells.
• The F plasmid replicates itself and transfers a copy of itself to the recipient bacterium
through the mating bridge.
• In some cases, chromosomal DNA from the donor bacterium may also be transferred
along with the F plasmid.
4) Recipient Cell:

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 • Upon receiving the F plasmid, the recipient bacterium becomes temporarily F-positive
(F+).
• The recipient cell can then act as a donor in subsequent conjugation events.
5) Integration:
•
The transferred F plasmid DNA can integrate into the recipient bacterium's genome,
converting it into an F+ cell.
• Alternatively, the plasmid may remain as a separate extrachromosomal element,
replicating independently in the recipient cell.
➢ Types of Conjugation
1) F+ × F- Conjugation:
• Occurs between an F-positive donor cell (containing the F plasmid) and an F-negative
recipient cell.
• The F plasmid is transferred from the donor to the recipient, converting the recipient
into an F+ cell.
2) Hfr Conjugation (High-frequency recombination):
• In some cases, the F plasmid integrates into the bacterial chromosome, creating an Hfr
(high-frequency recombination) donor cell.
• During conjugation, chromosomal DNA adjacent to the integrated F plasmid is
transferred along with the plasmid.
• This chromosomal DNA can recombine with the recipient cell's chromosome,
potentially transferring chromosomal genes.

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 Module-5
Concept of Sterilization
Sterilization is a critical process in various fields, including healthcare, food industry, and
laboratory work, aimed at eliminating or killing all forms of microbial life, including
bacteria, viruses, fungi, and spores. This process ensures that the risk of infection,
contamination, or spoilage is minimized or eliminated.

Physical methods of sterilization

1) Sun light - It is responsible for spontaneous sterilization in natural conditions. In tropical
countries, the sunlight is more effective in killing germs due to combination of ultraviolet
rays and heat. By killing bacteria suspended in water, sunlight provides natural method of
disinfection of tanks and lakes.
2) Drying - Moisture is essential for growth of bacteria. Drying in air has dangerous effect on
many bacteria. However, spores are unaffected. Therefore, it is not satisfactory method for
sterilization.
3) Heat sterilization - it is of two types:
A) Dry heat sterilization- In dry heat sterilization, dry heat is used for sterilizing different
materials. Heated air or fire is used in this process. As compared to the moist heat
sterilization, the temperature is higher. The temp. is maintained for almost an hour to kill
the most difficult of the resistant spores. Dry heat sterilization also have 4 type:-
I. Hot air oven
II. Red hot sterilization
III. Flaming
IV. Incineration
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 B) Moist heat sterilization: - Moist heat method is used for heat sensitive materials and
materials through which steam is permeable. culture media is also sterilized through moist
heat sterilization. It has also types: -
I. Below 100°C
II. Above 100°C
III. At 100°C
IV. Pasteurization

I. HOT AIR OVEN

Principle
• Sterilizing by dry heat is accomplished by conduction.
• The heat is absorbed by the outside surface of the item, then passes towards the centre of
the item, layer by layer.
• The entire item will eventually reach the temperature required for sterilization to take place.
• Dry heat does most of the damage by oxidizing molecules.
• The essential cell constituents are destroyed and the organism dies.
• The temperature is maintained for almost an hour to kill the most difficult of the resistant
spores.
➢ Generally, they can be operated from 50 to 300 °C, using a thermostat to control the
temperature, An air circulating fan helps in uniform distribution of the heat.
II. Red hot sterilization
Sterilization by holding them in Bunsen flame till they become red hot. It use for bacteriological
loops, straight wires, tips of forceps & spatulas
III. Flaming
This is a method of passing article over a flame, but not heating it to redness. Use- scalpels,
mouth of test tubes, flask, glass slide & cover slips .
IV. Incineration
Incineration is a waste treatment process that involves of organic substances contained in waste
materials. This method also burns any organism to ash. It is used to sterilize medical and other
biohazardous waste before it is discarded with non-hazardous waste.
➢ Moist Heat (or Steam): Uses hot water.
■ Moist heat kills microorganisms by denaturing proteins.
■Moist heat may be used in three forms to achieve microbial inactivation
1. Dry saturated steam - Autoclaving
2. Boiling water/ steam at atmospheric pressure
3. Hot water below boiling point
4. Pasteurization

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•Moist heat sterilization involves the use of steam in the range of 121-134°C.
•Steam under pressure is used to generate high temperature needed for sterilization.
•Saturated steam acts as an effective sterilizing agent.
•Steam for sterilization can be either wet saturated steam (containing entrained water droplets)
or dry saturated steam (no entrained water droplets).
Boiling (At 100 °C)
• Quite common especially in domestic circumstances.
• Boiling is done for metallic devices like surgical scissors, scalpels, needles etc like
instruments. Here substances are boiled to sterilize them.
Pasteurization (Below 100 °C)
• Pasteurization is the process of heating the milk at a temperature of 60 degrees or 72 degrees
3 to four times. Here alternative heating and cooling kills all the microbes and molds without
boiling the milk.
Autoclave (Above 100 °C)
• The air in the chamber is flushed out and filled with saturated steam.
• Water is boiled to produce steam, which is released through the jacket and into the
autoclave's chamber
• Hot, saturated steam enters the chamber and the desired temperature and pressure, usually
121°C
• At this temperature saturated steam destroys all vegetative cells and endospores.
• Moist heat is thought to kill so effectively by degrading nucleic acids and by denaturing
enzymes and other essential proteins.
• It also may disrupt cell membranes. The chamber is closed tightly the steam keeps on
filling into it and the pressure gradually increases.
• The items to be sterilized get completely surrounded by saturated steam (moist heat) which
on contact with the surface of material to be sterilized condenses to release its latent heat
of condensation which adds to already raised temperature of steam so that eventually all
the microorganisms in what ever form are killed.
• The usual temperature achieved is 121 °C at a pressure of 15 [Link]. at exposure time of
only 15-20 mins. By increasing the temperature, the time for sterilizing is further reduced.
RADIATION
Two types of radiation: Ionising radiation & non-ionising radiation

1) Non-ionising radiation (HOT STERILIZATION): Infrared- Used for rapid mass

sterilization of pre-packed items such as Syringe, Catheter’s UV Used for disinfecting
enclosed area such as entryways, operation theatres and labs.
2) Ionising radiation (Cold sterilization) Gamma rays: X-rays: Used for sterilising plastics,
syringes, swabs, catheters, animal feeds, cardboard, oils, greases, fabric and metal foils.
➢ MECHANISM
a) Non-ionizing radiations(UV light) Induce the production of abnormal nucleotides such as
thymine dimers in the bacterial cell.
b) lonizing radiation(X-rays, gamma rays,cathode rays) Produces microbial mutant Causes
ionization resulting in the death of cell.

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 Chemical methods of sterilization
Chemical methods of sterilization involve the use of chemical agents to eliminate or kill all
forms of microbial life, including bacteria, viruses, spores, and fungi. These methods are crucial
in medical settings, laboratories, and various industries where sterilization is essential for safety
and hygiene. Here are some common chemical sterilization methods:

1) Ethylene Oxide (EtO) Sterilization

• Mechanism: Ethylene oxide gas disrupts the DNA and proteins of microorganisms,
leading to cell death.
• Applications: Used for sterilizing medical devices, surgical instruments, and sensitive
equipment that cannot withstand high temperatures.
• Advantages: Effective for a wide range of materials, including plastics and electronics.
• Disadvantages: Toxicity and potential carcinogenic effects require careful handling
and aeration post-sterilization.

2) Hydrogen Peroxide (H2O2) Plasma Sterilization

• Mechanism: Hydrogen peroxide vapor, when energized by plasma, creates reactive
species that disrupt cell membranes, DNA, and proteins.
• Applications: Sterilizes heat-sensitive instruments, including endoscopes and certain
surgical tools.
• Advantages: Low temperature, environmentally friendly, and leaves no toxic residues.
• Disadvantages: Limited penetration and not suitable for materials that absorb moisture.

3) Formaldehyde Sterilization
• Mechanism: Formaldehyde gas alkylates microbial proteins and nucleic acids, leading
to cell death.
• Applications: Used in biological safety cabinets, for decontaminating rooms and
equipment.
• Advantages: Effective against a wide range of microorganisms.
• Disadvantages: Highly toxic and irritating to mucous membranes, requires thorough
aeration.

4) Glutaraldehyde Sterilization
• Mechanism: Glutaraldehyde acts by cross-linking microbial proteins and DNA,
leading to cell death.
• Applications: Sterilizes medical and dental instruments, especially those made of
materials sensitive to heat.
• Advantages: Broad spectrum of antimicrobial activity.
• Disadvantages: Toxicity requires careful handling and thorough rinsing of instruments.

5) Peracetic Acid Sterilization

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 • Mechanism: Peracetic acid oxidizes essential cell components, including proteins and
lipids, resulting in cell death.
• Applications: Sterilization of medical devices, endoscopes, and food processing
equipment.
• Advantages: Effective at low concentrations and temperatures, environmentally
friendly, leaves no toxic residues.
• Disadvantages: Corrosive to some metals and materials, requires proper handling and
storage.

6) Chlorine Dioxide Sterilization

• Mechanism: Chlorine dioxide gas or solution disrupts microbial cell walls and inhibits
cellular enzymes.
• Applications: Sterilizes medical equipment, rooms, and in water treatment.
• Advantages: Highly effective, even at low concentrations, and does not produce toxic
residues.
• Disadvantages: Corrosive, requires special handling and equipment for safe
application.

7) Ozone Sterilization
• Mechanism: Ozone gas oxidizes and destroys microbial cell components, including
DNA and proteins.
• Applications: Sterilization of medical devices, water treatment, and food processing.
• Advantages: Highly effective and environmentally friendly.
• Disadvantages: Highly reactive and potentially toxic, requires specialized equipment
and safety measures.

Disinfectants
Disinfectants are chemical agents used to reduce or eliminate harmful microorganisms from
surfaces and objects. Unlike sterilization, which aims to kill all microbial life, disinfection
typically targets pathogens to reduce the risk of infection. Disinfectants are widely used in
healthcare settings, food industries, households, and other environments to maintain hygiene
and prevent the spread of diseases. Here are some common types of disinfectants and their
characteristics:

1) Alcohols
• Examples: Ethanol (70%), Isopropanol (70-90%)
• Mechanism: Denatures proteins and dissolves lipids, effectively killing bacteria, fungi,
and some viruses.
• Applications: Disinfecting skin (hand sanitizers), surfaces, and medical instruments.
• Advantages: Rapid action, leaves no residue, relatively safe.

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 • Disadvantages: Flammable, evaporates quickly, ineffective against spores and some
viruses.

2) Chlorine and Chlorine Compounds

• Examples: Sodium hypochlorite (bleach), Chlorine dioxide
• Mechanism: Oxidizes cellular components, disrupting metabolic functions and leading
to cell death.
• Applications: Disinfecting water, surfaces, and medical equipment.
• Advantages: Broad-spectrum efficacy, inexpensive.
• Disadvantages: Corrosive, can produce toxic by-products, may cause irritation.

3) Hydrogen Peroxide
• Mechanism: Produces free radicals that damage cellular components, including lipids,
proteins, and DNA.
• Applications: Disinfecting surfaces, medical instruments, and contact lenses.
• Advantages: Effective against a broad range of microorganisms, environmentally
friendly (decomposes into water and oxygen).
• Disadvantages: Can be corrosive to some materials, may cause irritation.

4) Quaternary Ammonium Compounds (Quats)

• Examples: Benzalkonium chloride, Dodecylbenzene sulfonic acid
• Mechanism: Disrupts cell membranes, leading to leakage of cellular contents and cell
death.
• Applications: Disinfecting surfaces, floors, and equipment.
• Advantages: Non-corrosive, effective against a variety of microorganisms, long-
lasting activity.
• Disadvantages: Less effective against non-enveloped viruses and spores, can leave
residues.

5) Phenolics
• Examples: Phenol, Cresols, Chloroxylenol
• Mechanism: Denatures proteins and disrupts cell membranes.
• Applications: Disinfecting surfaces, equipment, and in some antiseptic formulations.
• Advantages: Effective against a wide range of microorganisms, including bacteria,
fungi, and viruses.
• Disadvantages: Can be toxic and irritating, may leave residues.

6) Aldehydes
• Examples: Glutaraldehyde, Formaldehyde

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 • Mechanism: Cross-links proteins and nucleic acids, leading to cell death.
• Applications: High-level disinfection of medical instruments and equipment.
• Advantages: Highly effective, broad-spectrum antimicrobial activity.
• Disadvantages: Toxicity and potential carcinogenic effects, requires careful handling
and thorough rinsing.

Definition of MIC
The Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of an
antimicrobial agent that will inhibit the visible growth of a microorganism after overnight
incubation. MIC is a critical measure in microbiology and pharmacology for determining the
effectiveness of antibiotics and other antimicrobial agents against specific bacteria, fungi, or
other pathogens.

Sanitization
Definition: Sanitization refers to the process of reducing microbial contamination on surfaces,
equipment, and environments to safe levels as determined by public health standards. It is less
comprehensive than sterilization and does not necessarily kill all microorganisms but
significantly reduces their numbers.
Applications:
• Food Industry: Cleaning food contact surfaces, utensils, and processing equipment.
• Public Spaces: Maintaining hygiene in restaurants, schools, and other communal areas.
• Healthcare: General cleaning of non-critical surfaces in hospitals and clinics.
Methods:
• Mechanical Cleaning: Using detergents and water to physically remove dirt and
microbes.
• Chemical Sanitizers: Using agents like chlorine compounds, quaternary ammonium
compounds, or alcohols.
• Heat: Using hot water or steam to sanitize surfaces and equipment.

Antisepsis
Definition: Antisepsis is the practice of using chemical agents (antiseptics) to inhibit or destroy
microorganisms on living tissues, such as skin and mucous membranes. The primary goal is
to prevent infection.
Applications:
• Healthcare: Pre-operative skin preparation, hand hygiene, wound cleaning.
• Personal Care: Use of antiseptic hand washes, mouthwashes, and topical applications.
Methods:
• Alcohols: Ethanol and isopropanol are common antiseptics used in hand sanitizers.
• Iodine Compounds: Povidone-iodine for skin disinfection.

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 • Chlorhexidine: Used in hand scrubs and as a pre-surgical skin antiseptic.
• Hydrogen Peroxide: Used for wound cleaning and mouth rinses.
• Quaternary Ammonium Compounds: Used in some antiseptic wipes and solutions.

Fumigation
Definition: Fumigation involves the use of chemical gases or vapors to disinfect an area by
killing or incapacitating pests and microorganisms. It is often used for large spaces or materials
that cannot be disinfected by other means.
Applications:
• Agriculture: Treating soil and stored grain to eliminate pests.
• Shipping and Warehousing: Disinfecting cargo holds, containers, and warehouses.
• Healthcare and Laboratories: Sterilizing rooms, equipment, and sensitive materials.
• Buildings: Eliminating pests such as termites, bedbugs, and rodents.
Methods:
• Chemical Gases:
o Methyl Bromide: Effective but heavily regulated due to environmental
concerns.
o Phosphine: Commonly used for grain storage and quarantine applications.
o Sulfuryl Fluoride: Used for structural fumigation against termites and other
pests.
o Ethylene Oxide: Used for sterilizing medical and pharmaceutical products.

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