1. A species is defined as a group of organisms that can reproduce and produce fertile offspring. Species are classified based on similar DNA, morphology, and ecology. Strains are genetic variants within a species. 2. Microorganisms are classified into six major types - bacteria, archaea, fungi, protozoa, algae, and viruses. They are identified using techniques like DNA sequencing, microscopy, and growth kinetics. 3. Microorganisms play important roles in ecosystems through decomposition, nutrient cycling, and symbiotic relationships. They are also used in industrial processes like fermentation which requires sterilization of equipment and growth media.

1. MODULE 9
Concept of species and strains:
Species is defined as the largest group of organisms in which any two individuals of
appropriate sexes can produce fertile offspring through sexual reproduction. Species
refer to organisms that are closely related.
In simple word, Species is a group of organisms that can reproduce with one another
in nature and produce fertile offspring.
Species is also a fundamental unit of classification. Species have similar
characteristics such as similar DNA sequences, morphological features, and
ecological features. Scientists consider “natural selection” as the origin of species.
Most importantly, the species that are originating from a common ancestor tend to
live in the same habitat. However, some have different breeds with great variation.
The total number of estimated species is between 8 to 8.7 million. However, only 14%
of these had been described till 2011. All species are given a two-part name
(binomial). The first name indicates the genus to which the species belongs. The
second name is the specific name or specific epithet. Furthermore, species were
observed from the time of Aristotle. Charles Darwin explained species could arise
from natural selection, and species may become extinct for a variety of reasons.
Strain - Strain is defined as a sub-type or a genetic variant of biological species.
Sometimes, it is called a culture of biological species. In microorganisms, strains tend
to originate from a single cell colony of microorganisms. It is often considered an
inherently artificial concept because it describes a specific intent such as genetic

2. isolation. Strains are commonly found in virology, botany, insects, and experimental
rodents.
Figure 02: Strains
For example, Influenza comprises four species: Influenza A, B, C, and D. These species
further divide into subtypes (strains) based on the viral proteins such as
haemagglutinin (H) and neuraminidase (N). The familiar subtypes (strains)
of Influenza A and Influenza B viral species are Influenza A (HINI), Influenza A
(H3N2), Influenza B (Victoria), Influenza B (Yamagata). Genetic mutations tend to bring
out the genetic variation in a strain. This process normally occurs during sexual
reproduction. Moreover, the other significant causes of genetic variation in a strain
are gene flow, crossing over between homologues chromosomes, random
fertilization, and random mating.
Identification and classification of microorganism:
Taxonomy is the classification, nomenclature and identification of microbes (algae,
protozoa, slime moulds, fungi, bacteria, archaea and viruses). The naming of organisms
by genus and species is governed by an international code.
Classification:
Classification is the orderly arrangement of bacteria into groups. There is nothing inherently
scientific about classification, and different groups of scientists may classify the same
organisms differently. For example, clinical microbiologists are interested in the serotype,
antimicrobial resistance pattern, and toxin and invasiveness factors in Escherichia coli,
whereas geneticists are concerned with specific mutations and plasmids.
Microorganisms or microbes are microscopic organisms that exist as unicellular,
multicellular, or cell clusters. Microorganims are widespread in nature and are beneficial to
life, but some can cause serious harm. They can be divided into six major types: bacteria,
archaea, fungi, protozoa, algae, and viruses

3. Bacteria:
Bacteria are unicellular organisms. The cells are described as prokaryotic because they lack a
nucleus. They exist in four major shapes: bacillus (rod shape), coccus (spherical shape),
spirilla (spiral shape), and vibrio (curved shape). Most bacteria have a peptidoglycan cell
wall; they divide by binary fission; and they may possess flagella for motility. The difference
in their cell wall structure is a major feature used in classifying these organisms.
According to the way their cell wall structure stains, bacteria can be classified as either
Gram-positive or Gram-negative when using the Gram staining. Bacteria can be further
divided based on their response to gaseous oxygen into the following groups: aerobic (living
in the presence of oxygen), anaerobic (living without oxygen), and facultative anaerobes (can
live in both environments).
According to the way they obtain energy, bacteria are classified as heterotrophs or
autotrophs. Autotrophs make their own food by using the energy of sunlight or chemical
reactions, in which case they are called chemoautotrophs. Heterotrophs obtain their energy by
consuming other organisms. Bacteria that use decaying life forms as a source of energy are
called saprophytes.
Archaea:
Archaea or Archaebacteria differ from true bacteria in their cell wall structure and lack
peptidoglycans. They are prokaryotic cells with avidity to extreme environmental conditions.
Based on their habitat, all Archaeans can be divided into the following groups: methanogens
(methane-producing organisms), halophiles (archaeans that live in salty environments),
thermophiles (archaeans that live at extremely hot temperatures), and psychrophiles (cold-
temperature Archaeans). Archaeans use different energy sources like hydrogen gas, carbon
dioxide, and sulphur. Some of them use sunlight to make energy, but not the same way plants
do. They absorb sunlight using their membrane pigment, bacteriorhodopsin. This reacts with
light, leading to the formation of the energy molecule adenosine triphosphate (ATP).
Fungi:
Fungi (mushroom, molds, and yeasts) are eukaryotic cells (with a true nucleus). Most fungi
are multicellular and their cell wall is composed of chitin. They obtain nutrients by absorbing
organic material from their environment (decomposers), through symbiotic relationships with
plants (symbionts), or harmful relationships with a host (parasites). They form characteristic
filamentous tubes called hyphae that help absorb material. The collection of hyphae is called
mycelium. Fungi reproduce by releasing spores.
Protozoa:
Protozoa are unicellular aerobic eukaryotes. They have a nucleus, complex organelles, and
obtain nourishment by absorption or ingestion through specialized structures. They make up
the largest group of organisms in the world in terms of numbers, biomass, and diversity.
Their cell walls are made up of cellulose. Protozoa have been traditionally divided based on
their mode of locomotion: flagellates produce their own food and use their whip-like

4. structure to propel forward, ciliates have tiny hair that beat to produce movement, amoeboids
have false feet or pseudopodia used for feeding and locomotion, and sporozoans are non-
motile. They also have different means of nutrition, which groups them as autotrophs or
heterotrophs.
Algae:
Algae, also called cyanobacteria or blue-green algae, are unicellular or multicellular
eukaryotes that obtain nourishment by photosynthesis. They live in water, damp soil, and
rocks and produce oxygen and carbohydrates used by other organisms. It is believed that
cyanobacteria are the origins of green land plants.
Viruses:
Viruses are noncellular entities that consist of a nucleic acid core (DNA or RNA) surrounded
by a protein coat. Although viruses are classified as microorganisms, they are not considered
living organisms. Viruses cannot reproduce outside a host cell and cannot metabolize on their
own. Viruses often infest prokaryotic and eukaryotic cells causing diseases.
Multicellular Animal Parasites:
A group of eukaryotic organisms consisting of the flatworms and roundworms, which
are collectively referred to as the helminths. Although they are not microorganisms
by definition, since they are large enough to be easily seen with the naked eye, they
live a part of their life cycle in microscopic form. Since the parasitic helminths are of
clinical importance, they are often discussed along with the other groups of
microbes.
Identification:
Identification is the practical use of classification criteria to distinguish certain organisms
from others, to verify the authenticity or utility of a strain or a particular reaction, or to isolate
and identify the organism that causes a disease.
Techniques used:
• DNA sequencing – to identify bacteria, moulds and yeasts.
• Riboprinter analysis – for bacterial identification and characterisation.
• Repeat–based polymerase chain reaction – for assessing the similarity of
microorganisms.
• Rapid pathogen confirmation by polymerase chain reaction.

5. Microscopy:
Microscopy is the technical field of using microscopes to view samples & objects that cannot be
seen with the unaided eye (objects that are not within the resolution range of the normal eye).
Used for:
Light microscopy is a general term used for any type of microscopy where light is being transmitted
from a source which is on the opposite side of the sample, to the objective lens. Generally, the light
is passed through a condenser to focus it on the sample to have maximum brightness. After the light
has passed through the sample, it goes through the objective lens to magnify the image of the
sample & then to the oculars, where the enlarged image is viewed.
Light microscopic techniques have significantly developed over the past 20 years & now provide an
indispensable tool to study molecular events at subcellular level in order to gain temporal & spatial
information at high resolution. To achieve optimal results, it is essential to carefully plan & carry
out microscopy-based experiments, which requires the understanding of at least the basics of
cell biology, sample preparation & fluorescence light microscopy.
Microscopists explore the relationships between structures & properties for a very wide variety of
materials ranging from soft to very hard, from inanimate materials to living organisms, in order to
better understand their behaviour.
Ecological aspects of single celled organisms:
Microorganisms have several vital roles in ecosystems: decomposition, oxygen
production, evolution, and symbiotic relationships. Decomposition is where
dead animal or plant matter is broken down into more basic molecules
Role in Ecosystems
Microorganisms such as bacteria, protozoa, viruses, and fungi play a large part in the

6. ecosystem. Discover the diverse ecological roles of microorganisms and their impacts on
ecosystems at large.
Sterilization and media compositions:
Media sterilization is the destruction or removal of all forms of microbial life
from the aqueous feedstock. In industrial fermentations, components such as
vessels, pipework, media, inlet air, and exhaust gases are frequently sterilized by a
combination of wet-heat and filtration methods.
Three primary methods of medical sterilization occur from high temperature/pressure
and chemical processes.
• Plasma Gas Sterilizers. ...
• Autoclaves. ...
• Vaporized Hydrogen Peroxide Sterilizers.
1. Plasma Gas Sterilizers:
Plasma sterilization utilizes low temperature hydrogen peroxide gas plasma within a
chamber to kill all living microorganisms on medical and dental equipment, including
bacteria, spores, viruses, and fungi. When vaporized hydrogen peroxide is added to the
chamber, equipment within the enclosure becomes sterile. Once the vapor is removed from
the chamber, a low temperature plasma is produced, ensuring complete sterilization for all
tools. The remnants of this process are water and oxygen, making operations safe for both
medical staff and the environment. Although plasma sterilization is a more expensive
method, it's highly effective and ideal for moisture sensitive medical tools.
2. Autoclaves:
An autoclave is a large, steel vessel or chamber that circulates steam at high temperature
and pressure to sterilize various items, or as part of an industrial process. Industrial
autoclave processes may include rubber vulcanization, composite parts processing, and
structural adhesive bonding. Autoclaves are one of the most economical methods of
sterilization, while still having a short cycle time.
3. Vaporized Hydrogen Peroxide Sterilizers:

7. Similar to plasma sterilization, vaporized hydrogen peroxide (VHP) sterilizers also utilize
hydrogen peroxide vapor, but plasma gas isn't used within the process. VHP sterilizers
remove humidity from an enclosure and hydrogen peroxide vapor is rapidly injected by a
generator to reach an effective concentration to sterilize equipment. These vapors
effectively remove micro-organisms that may be present, sterilizing the enclosure. The
generator then reverses the process, breaking down the hydrogen peroxide vapor into
environmentally friendly elements. This method has one of the lowest cycle times, resulting
in the ability to sterilize equipment in high volume batches.
Growth kinetics:
Growth kinetics is an autocatalytic reaction which implies that the rate of growth is directly
proportional to the concentration of cell. The cell concentration is measured by direct and
indirect methods.
Xo: initial biomass concentration (mg/ml)
Xmax: maximum biomass concentration (mg/ml)
DCW: dry cell weight
Μmax: maximum specific growth rate (h-1)
or
Growth kinetics is an autocatalytic reaction which implies that the rate of growth is directly
proportional to the concentration of cell. The cell concentration is measured by direct and indirect
methods.
Classified based on the relationship between product synthesis and energy generation in the cell:
• Growth associated
• Non-growth associated
• Mixed-growth associated
Growth associated -
Growth linked products are formed by growing cells and hence primary metabolites. Figure clearly
shows that product is formed simultaneously with growth of cells. That is product concentration
increases with cell concentration. The formation of growth associated product may be described
by Eq. (1);

8. dP / dt = rp= qpX (E1)
where P = concentration of product
qp = specific rate of product formation
X = biomass concentration.
Non-growth associated -
They are formed by cells which are not metabolically active and hence are called secondary
metabolites. Figure 2 clearly shows that product formation is unrelated to growth rate but is a
function of cell concentration. The formation of Non-growth associated product may be
described by Eq. (2);
Figure 2.
Non-growth associated.
Qp = β = constant ( E2)
Mixed-growth associated -
The product formation from the microorganism depends on both growth and Non-growth
associated. It takes place during growth and stationary phases. In Figure 3, product formation is a
combination of growth rate and cell concentration. The formation of Mixed-growth associated
product may be described by Eq. (3);

9. Figure 3.
Mixed growth associated.
Qp = αμ + β (E3)