Unit9: Microbiology

Microbiology: It is the branch of biology that deals with microorganisms.

Role of microbes in modern biotechnology

1. In antibiotic production
2. In production of dairy products
3. In food and beverages industry
4. In genetic engineering
5. In vaccine production

Characteristics of microorganisms
1. Microscopic organisms (In general required microscope for visualization)
2. They exist as unicellular(Bacteria, unicellular fungi: Yeast, unicellular algae:
Chlamydomonas), multicellular(fungi), or cell clusters
3. They are widespread in nature and are beneficial/detrimental to life
4. However, but some can cause serious harm (Pneumonia)
5. Microbes can be divided into six major types: bacteria, archaea, fungi,
protozoa, algae, and viruses.
6. Microorganisms can be prokaryotes or eukaryotes

Features of unicellular organism

1. An unicellular organism is an organism that exists throughout their entire
life cycle as a single cell
2. All life processes such as reproduction, feeding, digestion, and excretion,
occur in one cell
3. They also known as single-celled organism
4. The unicellular organisms usually reproduce by asexual means (cellular
fission/Cell division)
5. They can be eukaryotes or prokaryotes
6. They possess whip-like structures (flagella) for movement
Difference between unicellular and multicellular organisms

Classification of Single-celled organisms

Structure of a typical bacterial cell

One of the distinct features of the bacterial cell is the presence of the cell wall
around the bacteria. Bacterial cell wall is of two types:
Classification methods of the microorganisms

Phenotypic method:

Morphological classification:Depending on their shape, bacteria are

classified into several varieties
1. Cocci (from kokkos meaning berry) are spherical or oval cells
2. Bacilli (from baculus meaning rod) are rod shaped cells
3. Vibrios are comma shaped curved rods and derive their name from
their characteristics vibratory motility
4. Spirilla are rigid spiral forms.
Based on composition of the cell wall
The Gram stain characterizes bacteria based on the structural characteristics of
their cell walls. The thick layers of peptidoglycan in the “Gram-positive” cell wall
stain purple, while the thin “Gram-negative” cell wall appears pink. Based on the
gram staining bacteria can be classified as “Gram positive” and “Gram negative”.

Genotypic method:

DNA-DNA hybridization
DNA–DNA hybridization measures the degree of genetic similarity between pools
of DNA sequences. It is usually used to determine the genetic distance between
two organisms. DNA–DNA hybridization was once used as a primary method to
distinguish bacterial species; a similarity value greater than 70% and ≤ 5 ºC in ΔTm
(difference in melting temperature) in the stability of the heteroduplex is
described as indicating that the compared strains belonged to distinct species. In
2014, a threshold of 79% similarity has been suggested to separate bacterial
subspecies.
%GC content
The genomic DNA G+C content, defined as the proportion of cytosines and guanines
within the overall number of nucleotides in the genome, is one of the most
frequently used features in taxonomic descriptions of microorganisms.Given the
rapid progress in sequencing technology along with some large-scale
genome-sequencing projects devoted exclusively to type strains , it is nowadays
reasonable to estimate the G+C content directly from completely sequenced
genomes.

Phylogenetic method

16S rRNA based analysis

16S rRNA gene sequencing is commonly used for identification, classification and
quantitation of microbes within complex biological mixtures such as environmental
samples. The 16S rRNA gene is a highly conserved component of the transcriptional
machinery of all DNA-based life forms and thus is highly suited as a target gene
for sequencing DNA in samples containing up to thousands of different species.
The 16S rRNA gene consists of both conserved and variable regions. While the
conserved region makes PCR amplification possible, sequencing the variable regions
allows discrimination between specific different microorganisms such as bacteria,
archaea and microbial eukarya.

Concept of species and Strain

A species is a closely related group of organisms with similar characteristics and
interbreed to produce a fertile offspring.
A species has a genetic similarity sufficient for mating, producing a fertile
offspring.

A strain is a genetic variant, subtype or culture of a biological species.

A strain is an isolate of a given species with a specific genetic characteristic, while
organisms in
Microscopy:

Microscopy: Microscopy is the technical field of using microscopes to view objects

and areas of objects that cannot be seen with the naked eye.

Types of Microscopy:

Microscope: A microscope is an instrument that can be used to examine objects

that are too small to be seen by the naked eye. Enlargement of the object is the
primary requirement for the examination/ to observe

Magnification: Magnification is the process of enlarging the apparent size (i.e.,

image), not physical size, of the object. The image of an object is magnified
through a set of lenses
When parallel rays pass through a convex lens it gets converged at a point called
focal length (f) of the lens. A lens with shorter focal length will have higher
magnification power.
Magnification is measured by multiples, such as 2x, 4x and 10x. In compound
microscope it will be i.e 10X, f=16mm; 40X, f=4 mm; 100X, f= 1.8mm
The overall magnification is given as the product of the magnification power of the
objective and ocular lenses (components of the compound optical microscope) and
the distance over which the image is projected:

Total Magnification = (250 *M1 * M2)/ 250 mm

Where, D = projection (tube) length (usually = 250 mm); M1, M2 = magnification of

objective and ocular. 250 mm = minimum distance of distinct vision for 20/20 eyes

Resolution: Resolution is the ability of any optical instruments to distinguish two

objects from each other

The limit of resolution is the closest distance between two points at which the
points still can be distinguished as separate entities. In the case of light
microscope the limit of resolution is 0.2μm
Magnification should be coupled with good resolution to visualize small
microorganisms, else magnification alone will produce an inconclusive or blurred
image
Maximum resolution or resolution limit = (0.061λ)/ N.A
λ= wavelength of light, N.A. = Numerical Aperture of lense (is a measure of the
light gathering capabilities of an objective lens).
In Microscope the two terms are important: Magnification and Resolution

Types of microscopes according to the source of illumination:

Illumination source: Light
1. Optical microscope: Optical microscope uses visible light as a source of
illumination. Light microscopes are able to magnify to a maximum of 1500X
and the limit of resolution of about 0.2 μm. Light microscope can be of two
types: Simple microscope (consisting of a single lens) and compound
microscope (consists of more than one lens).
2. Dark field microscope
3. Phase contrast microscope
4. Fluorescence microscope
5. Confocal microscope
 Components of a Compound Optical Microscope:

Illumination source: Electron beam

Electron Microscope:
The main differences between electron microscope and light microscope are:
a. Electron microscope use electromagnetic lenses instead of optical lenses
b. It uses beam of electron as a source of illumination
As the wavelength of an electron can be up to 100,000 times shorter than that of
visible light photons, electron microscopes have a higher resolving power (low
resolution limit) than light microscopes and can reveal the structure of smaller
objects.
There are two types of Electron microscope
1. Transmission Electron microscope (TEM)
2. Scanning Electron microscope (SEM)
Microbial Culture media

Culture media is a liquid or solid or semi-solid combination of nutrients, water, pH

buffer that supports growth of microbes under laboratory condition.

Classification of Culture media

1. According to the consistency
a. Liquid
b. Solid: Contains 1.5-2.0% agar as a solidifying agent
c. Semi-solid: Contains 0.5% agar as a solidifying agent
2. According to the composition
a. Defined media: A defined medium will have known quantities of all
ingredients.
b. Undefined media: An undefined medium has some complex
ingredients, such as yeast extract, which consists of a mixture of
many, many chemical species in unknown proportions.
3.According to the application

a. Nutrient media –This is an undefined medium. These media contain all

the elements that most bacteria need for growth and are
non-selective, so they are used for the general cultivation and
maintenance of bacteria kept in laboratory-culture collections.
b. Minimal media – Media that contains the minimum nutrients possible
for colony growth, generally without the presence of amino acids, and
are often used by microbiologists and geneticists to grow “wild type”
microorganisms.
c. Selective media – Used for the growth of only selected
microorganisms.
d. Differential media – Also known as indicator media, are used to
distinguish one microorganism type from another growing on the same
media.
The concept of sterilization

Sterilization refers to any process that removes, kills, or deactivates all forms of
life from a specific surface, object or fluid.

When we are interested in growing a particular microbe(e.g., bacteria) we need to

remove other unwanted microbes from the culture media. Sterilization in
microbiology is hence a process that removes, kills, or deactivates microorganisms
from the culture media before the beginning of any microbiological work

Terminologies used in Sterilization

Decontamination: Decontamination is the process of removing contaminants on an

object or area, including chemicals, micro-organisms or radioactive substances.
This may be achieved by chemical reaction, disinfection or physical removal

Disinfection: Disinfection is the process, which involves the elimination of most

pathogenic microorganisms (excluding bacterial spores) on inanimate objects.

Antisepsis: Prevention of infection by inhibiting or arresting the growth and

multiplication of germs (infectious agents).

Bactericidal agents/germicides: Those which are able to kill bacteria.

Bacteriostatic agents: Those which are only able to prevent the growth of the
bacteria but unable to kill them.

Difference between Sterilization and Disinfection

Disinfection Sterilization

In this, the number of harmful In this, the medium is made completely

microbes is minimized to a negligible free from all microbes.
level.
 It kills only vegetative cells and not It kills both vegetative cells and spores.
the spores.

Wounds are disinfected – with agents Wounds cannot be sterilized – as it may

such as hydrogen peroxide or rubbing kill surrounding healthy cells.
alcohol.

Disinfection only reduces the effect Sterilization completely rids microbes

of microbes. from the surface

Chemical methods are used for Combination of heat, irradiation, high

disinfection pressure, chemical and physical methods
are used for sterilization

Importance of sterilization

1. To prevent contamination in sterile products

2. To prevent transmission of pathogenic microorganisms which are responsible
for causing disease
3. To prevent decomposition and spoilage of food products
4. To prevent unwanted microbial contamination in pure culture used in
microbiological experiments, in antibiotics, enzyme, fermentation and other
industrial processes

Types of Sterilization
Commonly used Sterilization method

1. Autoclaving: Culture vessels, culture media are generally sterilized by this

method in an instrument commonly known as autoclave and the method is
known as autoclaving. In this process the steilizable objects are treated
with steam at 121oC temperature and 15 psi pressure for 15-20 mins in a
closed vessels (autoclave)
2. Flame sterilization: Instruments like needles, forceps are sterilized during
the work by dipping them into the 95% alcohol followed by flaming (oxidizing
flame)

Microbial/Bacterial Growth Kinetics

Doubling/generation time: The doubling time is time it takes for a population to

double in size/value

Bacterial Growth Curve:

The bacterial growth curve represents the number of live cells in a bacterial
population over a period of time.

Lag Phase: This initial phase is characterized by cellular activity but not
growth.
Exponential (Log) Phase: After the lag phase, bacterial cells enter the
exponential or log phase. This is the time when the cells are divided by
binary fission and doubling in numbers after each generation time. Metabolic
activity is high as DNA, RNA, cell wall components, and other substances
necessary for growth are generated for division.
Stationary Phase: Eventually, the population growth experienced in the log
phase begins to decline as the available nutrients become depleted and waste
products start to accumulate. Bacterial cell growth reaches a plateau, or
stationary phase, where the number of dividing cells equal the number of
dying cells.
Death Phase: As nutrients become less available and waste products
increase, the number of dying cells continues to rise. In the death phase, the
number of living cells decreases exponentially and population growth
experiences a sharp decline. As dying cells lyse or break open, they spill their
contents into the environment making these nutrients available to other
bacteria. This helps spore producing bacteria to survive long enough for
spore production.