Centrifugation water from your clothes, and blood

Centrifugation is the process where a mixture is separated
through spinning. It is used to separate skim milk from whole milk, water from
your clothes, and blood cells from your blood plasma. Although centrifugation
is primarily used to separate mixtures, it is also used to test the effects of
gravity on people and objects. While for DNA extraction we use this technique
for many microorganisms including bacteria and fungi.

Gel Electrophoresis

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Gel electrophoresis is a method for separation and analysis
of macromolecules (DNA, RNA and proteins) and their fragments, based on their
size and charge. It is used in clinical chemistry to separate proteins by
charge and/or size (IEF agarose, essentially size independent) and in
biochemistry and molecular biology to separate a mixed population of DNA and
RNA fragments by length, to estimate the size of DNA and RNA fragments or to
separate proteins by charge.

Nucleic acid molecules are separated by applying an electric
field to move the negatively charged molecules through a matrix of agarose or
other substances. Shorter molecules move faster and migrate farther than longer
ones because shorter molecules migrate more easily through the pores of the gel.
This phenomenon is called sieving. Proteins are separated by charge in agarose
because the pores of the gel are too large to sieve proteins. Gel
electrophoresis can also be used for separation of nanoparticles.

Gel electrophoresis uses a gel as an antconvective medium
and/or sieving medium during electrophoresis, the movement of a charged
particle in an electrical field. Gels suppress the thermal convection caused by
application of the electric field, and can also act as a sieving medium,
retarding the passage of molecules; gels can also simply serve to maintain the
finished separation, so that a post electrophoresis stain can be applied. DNA
Gel electrophoresis is usually performed for analytical purposes, often after
amplification of DNA via polymerase chain reaction (PCR), but may be used as a
preparative technique prior to use of other methods such as mass spectrometry,
RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further
characterization.

PCR

Polymerase chain reaction (PCR) is a technique used in
molecular biology to amplify a single copy or a few copies of a segment of DNA
across several orders of magnitude, generating thousands to millions of copies
of a particular DNA sequence. PCR is probably the most widely used technique in
molecular biology. This technique is used in biomedical research, criminal
forensics, and molecular archaeology.

Plating
methods

In the laboratory various plating
methods are used for isolation, propagation and enumeration of microorganisms.
All methods uses aseptic techniques in order to maintain sterile environment.

Plating methods includes the
following;

Streak-plate
method
Pour-plate
 method
Spread-plate
 method
Soft agar
overlays method
Replica-plate
method

Streak plate method:

It is used for isolation of pure
culture of bacteria from mixed culture. In a colony millions of bacterial cells

In this method the sample is
spread over the agar plates with the help of sterile metal loop in such a way
that the bacteria can deposit over the plates separately and upon incubation
they develop into colonies.   Before
streaking the loop is sterilized by passing it on a flame and is cooled before
picking of inoculum. The streaking is performed in a zigzag manner. .The plate contain more growth in the first
section. The second section have less growth, while the final section possess
least amount of growth and the colonies were isolated.

Pour plate
method:

This method is used for determination of
microorganisms in a liquid sample. In this method 1ml of inoculum is poured in
the center of petri plate with the help of sterile pipette. 5ml of molten
cooled agar is then poured into the plate containing the sample or inoculum and
is mixed well. After solidification the plates were placed in the incubator for
24-48 hours.

Spread plate
procedure:

This method is used for quantification of
bacteria in a solution. In this method a glass or metal spreader is used to
apply small amount of suspended bacteria in a solution over a plate. Like pour
plate method serial dilutions are made for countable plate. 1ml of sample is
poured from the dilution series onto the agar plates. The sample is then spread
evenly with the sterilized L shaped spreader. The plates were then incubated at
37C for 24-48 hours. CFU value of the sample is calculated. After counting the
colonies multiply with the dilution factor

Soft agar
overlays method:

This is used for the detection of
bacteriophages that range in size from 100-200 nm. Phages require bacterial
cells as a host for replication, therefor their propagation require mixture of
host cells and phages. In this method 50 ?l to 200 ?l, of a phage suspension is
placed in a tube containing about 108 bacteria which is then spread on molten
nutrient agar. Then this mixture is placed on the surface of hard nutrient agar
plate. The plates were placed in incubator and after some time cloudy
appearance of plate indicates growth of bacteria. Formation of plaques
indicates that phages are present and they infect the bacteria and cause their
lysis.

Replica plate
method:

This method allows the comparison between
primary and secondary plates. This is useful for phenotype selection. First
primary plate is made by spread plate method. Secondary plate contain growth
inhibitors or it lack a particular nutrient. Secondary plate is inoculated with
the cells from primary one. A piece of velvet is pressed on a primary plate so
that the bacterial cells adhere to it and is then transferred to various
secondary plates. This allows screening of phenotypes in a single experiment.

 

 

 

 

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