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Microbiology is the study of microorganisms including viruses, bacteria, fungi,

and protozoa.

- Microbiological aspects are very important to humans because many diseases are caused

by microorganisms.

- Moreover, microbiology also helps to develop many industrial applications with the help

of microorganisms, for example; bakery industry, pharmaceutical industry, beer industry,

etc.

Two visions of microbiology

(1) pure microbiology, which includes bacteriology, mycology, protozoology,

parasitology, immunology, virology, etc.

(2) applied microbiology which include medical microbiology, pharmaceutical

microbiology, industrial microbiology, food microbiology etc.

Microbiological use various equipment like microscopes and various dyes and stains, and all

these equipment must be sterile. Many microbiological techniques like agar diffusion test,

ATP test, bacterial inhibition assay, CAMP test, endospore staining, indole test,

microbiological culture, etc. are used in Microbiology.

1. Microbiology

2021-10-01 KNU Microbial Biotechnology Laboratory 2

미생물학(microbiology)의 종류

* 미생물학: 미생물을 연구하는 학문

-미생물 형태, 구조, 이용, 생태, 생리, 분류, 영양, 작용, 대사, 유전 등

가. 연구내용에 따라

1) 일반미생물학(General microbiology)

: 미생물의 이론적 기초연구, 미생물에 대한 기본원리 연구

= 기초미생물학(Basic microbiology)

= 이론미생물학(Theoretical microbiology)

= 순수미생물학(Pure microbiology)

2) 응용미생물학(Applied microbiology): 미생물의 응용에 관한 연구

= 산업미생물학 = 공업미생물학(Industrial microbiology)

= 발효미생물학(Fermentation microbiology)

* 농업미생물학(Agricultural microbiology)

3) 병원미생물학(Pathogenic microbiology): 동식물의 병원균에 관한 연구

= 의학미생물 + 수의미생물 + 식물 병원미생물

* 보건미생물학(public health microbiology) : 인체건강과 관련된 미생물 연구


나. 생태적 특징에 따라

1) 식품미생물학(food microbiology): 식품 관련 미생물 연구

2) 토양미생물학(Soil microbiology): 토양 자정작용, 농업 이용

3) 환경미생물학(Environmental microbiology)

-폐수처리, 난분해성 및 독성물질 분해, 생물복원 등

4) 낙농미생물학(Dairy microbiology): 유제품의 가공, 저장

5) 해양미생물학(Marine microbiology): 해양의 자정작용

* 우주미생물학(Space microbiology)

대기미생물학(Air microbiology)

수생미생물학(Aquatic microbiology)

다. 분류에 따라

1) 세균학(Bacteriology): 세균을 주로 연구

2) 균류학(Mycology): 효모, 곰팡이(버섯 포함) 주로 연구

3) 원생동물학(Protozoology): 원생동물 연구

4) 조류학(Algology): 담수나 해수의 조류 연구

5) 바이러스학(Virology): 동물 및 식물 병원성 virus 연구


Biology is the scientific study of life. It is a natural science with a broad scope but has

several unifying themes that tie it together as a single, coherent field.

1) For instance, all organisms are made up of cells that process hereditary information

encoded in genes, which can be transmitted to future generations.

2) Another major theme is evolution, which explains the unity and diversity of life.

3) Finally, all organisms require energy to move, grow, and reproduce, as well as to regulate

their own internal environment.

Biologists are able to study life at multiple levels of organization. From the molecular

biology of a cell to the anatomy and physiology of plants and animals, and evolution

of populations.

Hence, there are multiple subdisciplines within biology, each defined by the nature of

their research questions and the tools that they use. Like other scientists, biologists use

the scientific method to make observations, pose questions, generate hypotheses,

perform experiments, and form conclusions about the world around them.

Life on Earth, which emerged more than 3.7 billion years ago, is immensely diverse.

Biologists have sought to study and classify the various forms of life,

from prokaryotic organisms such as archaea and bacteria to eukaryotic organisms such

as protists, fungi, plants, and animals. These various organisms contribute to

the biodiversity of an ecosystem, where they play specialized roles in

the cycling of nutrients and energy through the biophysical environment.

Biology


Cell biology (also cellular biology or cytology) is a branch of biology that studies the

structure, function and behavior of cells. Cell biology encompasses

both prokaryotic and eukaryotic cells and can be divided into many sub-topics which may

include the study of cell metabolism, cell communication, cell cycle, biochemistry,

and cell composition.

The study of cells is performed using several techniques such as cell culture, various types

of microscopy, and cell fractionation. These have allowed for and are currently being used

for discoveries and research pertaining to how cells function, ultimately giving insight

into understanding larger organisms.

Knowing the components of cells and how cells work is fundamental to all biological

sciences while also being essential for research in biomedical fields such as cancer, and

other diseases. Research in cell biology is interconnected to other fields such

as genetics, molecular genetics, biochemistry, molecular biology, medical

microbiology, immunology, and cytochemistry.

Modern-day cell biology research looks at different ways to culture and manipulate cells

outside of a living body to further research in human anatomy and physiology, and to

derive medications. The techniques by which cells are studied have evolved. Due to

advancements in microscopy, techniques and technology have allowed for scientists to

hold a better understanding of the structure and function of cells.

Cell Biology


Molecular biology is the branch of biology that concerns the molecular basis of biological

activity in and between cells, including molecular synthesis, modification, mechanisms

and interactions. The central dogma of molecular biology describes the process in which

DNA is transcribed into RNA, which is then translated into protein.

William Astbury described molecular biology in 1961 in Nature, as:

...not so much a technique as an approach, an approach from the viewpoint of the so-called

basic sciences with the leading idea of searching below the large-scale manifestations of

classical biology for the corresponding molecular plan. It is concerned particularly with

the forms of biological molecules and [...] is predominantly three-dimensional and

structural – which does not mean, however, that it is merely a refinement of morphology.

It must at the same time inquire into genesis and function.

Some clinical research and medical therapies arising from molecular biology are covered

under gene therapy whereas the use of molecular biology or molecular cell biology in

medicine is now referred to as molecular medicine. Molecular biology also plays

important role in understanding formations, actions, and regulations of various parts

of cells which can be used to efficiently target new drugs, diagnose disease, and

understand the physiology of the cell.

Molecular Biology


What is life?


BIOLOGY CELL BIOLOGY

MOLECULAR BIOLOGY

Molecular microbiology deals with molecular mechanisms and physiological processes of

microbes and their utilization in production of biotechnology products and medicines such

as vaccines, antibodies. It also involves advancement in pathogenicity of microbes.

A microbiologist studies the different characteristics of microscopic organisms to identify

aspects such as growth and development. A molecular microbiologist studies the small

parts that make up these organisms to identify how microorganisms, such as parasites,

bacteria, and viruses, interact with their hosts.

The key difference between molecular microbiology and molecular biology is that

molecular microbiology is the study of microorganisms at molecular level

whereas molecular biology is the study of biological activities at molecular level.

Molecular microbiology is the fastest growing discipline which plays significant role for the

detection and characterization of microorganism. Rapid detection of microorganism by

using these molecular methods has revolutionized routine diagnostic microbiology.

Microbiology can be tough if you don't know your basic molecular biology. Especially

when thinking about how organisms move, grow and infect other living things. That's

usually why molecular biology comes before microbiology in a science-based curriculum

(especially med school!).

Molecular Microbiology


MICROBIOLOGY MOLECULAR MICROBIOLOGY

2.1 Molecular cloning

2.2 Polymerase chain reaction

2.3 Gel electrophoresis

2.4 Macromolecule blotting and probing

2.4.1 Southern blotting

2.4.2 Northern blotting

2.4.3 Western blotting

2.4.4 Eastern blotting

2.5 Microarrays

2.6 Allele-specific oligonucleotide

2. Techniques in molecular microbiolgy


One of the most basic techniques of molecular biology to study protein function

is molecular cloning.

In this technique, DNA coding for a protein of interest is cloned using polymerase chain

reaction (PCR), and/or restriction enzymes into a plasmid (expression vector). A vector

has 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a

selective marker usually antibiotic resistance.

Located upstream of the multiple cloning site are the promoter regions and

the transcription start site which regulate the expression of cloned gene. This plasmid can

be inserted into either bacterial or animal cells.

Introducing DNA into bacterial cells can be done by transformation via uptake of naked

DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing

DNA into eukaryotic cells, such as animal cells, by physical or chemical means is

called transfection.

Several different transfection techniques are available, such as calcium phosphate

transfection, electroporation, microinjection and liposome transfection. The plasmid may

be integrated into the genome, resulting in a stable transfection, or may remain

independent of the genome, called transient transfection.

2-1. Molecular cloning


Procedures


Transformation


Transduction


Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA.

In brief, PCR allows a specific DNA sequence to be copied or modified in predetermined

ways. The reaction is extremely powerful and under perfect conditions could amplify one

DNA molecule to become 1.07 billion molecules in less than two hours.

The PCR technique can be used to introduce restriction enzyme sites to ends of DNA

molecules, or to mutate particular bases of DNA, the latter is a method referred to as site-

directed mutagenesis.

PCR can also be used to determine whether a particular DNA fragment is found in a cDNA

library.

PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of

RNA, and, more recently, quantitative PCR which allow for quantitative measurement of

DNA or RNA molecules.

2-2. Polymerase Chain Rection (PCR)


PCR primer


PCR의 과정




Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is

that DNA, RNA, and proteins can all be separated by means of an electric field and size.

In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by

running the DNA through an electrically charged agarose gel.

Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of

size and their electric charge by using what is known as a 2D gel electrophoresis.

2-3. Gel electrophoresis


Agarose gel 전기영동


PAGE 전기영동


The terms northern, western and eastern blotting are derived from what initially was a

molecular biology joke that played on the term Southern blotting, after the technique

described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas,

developer of the RNA blot which then became known as the northern blot, actually didn't

use the term.

1. Southern blotting

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for

probing for the presence of a specific DNA sequence within a DNA sample.

DNA samples before or after restriction enzyme (restriction endonuclease) digestion are

separated by gel electrophoresis and then transferred to a membrane by blotting

via capillary action.

The membrane is then exposed to a labeled DNA probe that has a complement base

sequence to the sequence on the DNA of interest.

Southern blotting is less commonly used in laboratory science due to the capacity of other

techniques, such as PCR, to detect specific DNA sequences from DNA samples.

2-4. Macromolecule blotting and probing



2. Northern blot

The northern blot is used to study the presence of specific RNA molecules as relative

comparison among a set of different samples of RNA.

It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this

process, RNA is separated based on size and is then transferred to a membrane that is then

probed with a labeled complement of a sequence of interest.

The results may be visualized through a variety of ways depending on the label used;

however, most result in the revelation of bands representing the sizes of the RNA detected

in sample.

The intensity of these bands is related to the amount of the target RNA in the samples

analyzed.

The procedure is commonly used to study when and how much gene expression is occurring

by measuring how much of that RNA is present in different samples, assuming that no

post-transcriptional regulation occurs and that the levels of mRNA reflect proportional

levels of the corresponding protein being produced.

It is one of the most basic tools for determining at what time, and under what conditions,

certain genes are expressed in living tissues.



3. Western blotting

In western blotting, proteins are first separated by size, in a thin gel sandwiched between

two glass plates in a technique known as SDS-PAGE. The proteins in the gel are then

transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support

membrane.

This membrane can then be probed with solutions of antibodies. Antibodies that specifically

bind to the protein of interest can then be visualized by a variety of techniques, including

colored products, chemiluminescence, or autoradiography. Often, the antibodies are

labeled with enzymes.

When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using

western blotting techniques allows not only detection but also quantitative analysis.

Analogous methods to western blotting can be used to directly stain specific proteins in

live cells or tissue sections.




4. Eastern blotting

The eastern blotting technique is used to detect post-translational modification of proteins.

Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications

using specific substrates.

It is a biochemical technique used to analyze protein post-translational

modifications including the addition of lipids, phosphates, and glycoconjugates. It is most

often used to detect carbohydrate epitopes. Thus, eastern blot can be considered an

extension of the biochemical technique of western blot.

Multiple techniques have been described by the term "eastern blot(ting)", most use prospo

protein blotted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-

PAGE) gel on to a polyvinylidene fluoride or nitrocellulose membrane.

Transferred proteins are analyzed for post-translational modifications using probes that may

detect lipids, carbohydrate, phosphorylation or any other protein modification.

Eastern blotting should be used to refer to methods that detect their targets through specific

interaction of the post-translational modifications and the probe, distinguishing them from

a standard far-western blot. In principle, eastern blotting is similar to lectin blotting (i.e.,

detection of carbohydrate epitopes on proteins or lipids).


A DNA microarray is a collection of spots attached to a solid support such as a microscope

slide where each spot contains one or more single-stranded

DNA oligonucleotide fragments.

Arrays make it possible to put down large quantities of very small (100 micrometre

diameter) spots on a single slide. Each spot has a DNA fragment molecule that is

complementary to a single DNA sequence. A variation of this technique allows the gene

expression of an organism at a particular stage in development to be qualified (expression

profiling).

In this technique the RNA in a tissue is isolated and converted to labeled complementary

DNA (cDNA). This cDNA is then hybridized to the fragments on the array and

visualization of the hybridization can be done.

Since multiple arrays can be made with exactly the same position of fragments, they are

particularly useful for comparing the gene expression of two different tissues, such as a

healthy and cancerous tissue. Also, one can measure what genes are expressed and how

that expression changes with time or with other factors.

There are many different ways to fabricate microarrays; the most common are silicon chips,

microscope slides with spots of ~100 micrometre diameter, custom arrays, and arrays with

larger spots on porous membranes (macroarrays).

There can be anywhere from 100 spots to more than 10,000 on a given array. Arrays can

also be made with molecules other than DNA.

2-5. Microarray





An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary

to the sequence of a variable target DNA. It acts as a probe for the presence of the target

in a Southern blot assay or, more commonly, in the simpler Dot blot assay. It is a common

tool used in genetic testing, forensics, and molecular Biology research.

An ASO is typically an oligonucleotide of 15–21 nucleotide bases in length. It is designed

in a way that makes it specific for only one version, or allele, of the DNA being tested.

Short (20–25 nucleotides in length), labeled probes are exposed to the non-fragmented

target DNA, hybridization occurs with high specificity due to the short length of the

probes and even a single base change will hinder hybridization. The target DNA is then

washed and the labeled probes that didn't hybridize are removed. The target DNA is then

analyzed for the presence of the probe via radioactivity or fluorescence.

The length of the ASO, which strand it is chosen from, and the conditions by which it

is bound to (and washed from) the target DNA all play a role in its specificity. These

probes can usually be designed to detect a difference of as little as 1 base in the target's

genetic sequence, a basic ability in the assay of single-nucleotide polymorphisms (SNPs),

important in genotype analysis and the Human Genome Project.

To be detected after it has bound to its target, the ASO must be labeled with a radioactive,

enzymatic, or fluorescent tag. The Illumina Methylation Assay technology takes

advantage of ASO to detect one base pair difference (cytosine versus thymine) to measure

methylation at a specific CpG site.

2-6. Allele-specific oligonucleotide



In molecular biology, procedures and technologies are continually being developed and

older technologies abandoned.

For example, before the advent of DNA gel electrophoresis (agarose or polyacrylamide),

the size of DNA molecules was typically determined by rate sedimentation in sucrose

gradients, a slow and labor-intensive technique requiring expensive instrumentation.

Aside from their historical interest, it is often worth knowing about older technology, as it is

occasionally useful to solve another new problem for which the newer technique is

inappropriate.

2-7. Others


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