Centrifugation is the process where a mixture is separatedthrough spinning. It is used to separate skim milk from whole milk, water fromyour clothes, and blood cells from your blood plasma. Although centrifugationis primarily used to separate mixtures, it is also used to test the effects ofgravity on people and objects. While for DNA extraction we use this techniquefor many microorganisms including bacteria and fungi.
Gel ElectrophoresisGel electrophoresis is a method for separation and analysisof macromolecules (DNA, RNA and proteins) and their fragments, based on theirsize and charge. It is used in clinical chemistry to separate proteins bycharge and/or size (IEF agarose, essentially size independent) and inbiochemistry and molecular biology to separate a mixed population of DNA andRNA fragments by length, to estimate the size of DNA and RNA fragments or toseparate proteins by charge.Nucleic acid molecules are separated by applying an electricfield to move the negatively charged molecules through a matrix of agarose orother substances. Shorter molecules move faster and migrate farther than longerones because shorter molecules migrate more easily through the pores of the gel.This phenomenon is called sieving. Proteins are separated by charge in agarosebecause the pores of the gel are too large to sieve proteins. Gelelectrophoresis can also be used for separation of nanoparticles.Gel electrophoresis uses a gel as an antconvective mediumand/or sieving medium during electrophoresis, the movement of a chargedparticle in an electrical field.
Gels suppress the thermal convection caused byapplication of the electric field, and can also act as a sieving medium,retarding the passage of molecules; gels can also simply serve to maintain thefinished separation, so that a post electrophoresis stain can be applied. DNAGel electrophoresis is usually performed for analytical purposes, often afteramplification of DNA via polymerase chain reaction (PCR), but may be used as apreparative technique prior to use of other methods such as mass spectrometry,RFLP, PCR, cloning, DNA sequencing, or Southern blotting for furthercharacterization.PCRPolymerase chain reaction (PCR) is a technique used inmolecular biology to amplify a single copy or a few copies of a segment of DNAacross several orders of magnitude, generating thousands to millions of copiesof a particular DNA sequence.
PCR is probably the most widely used technique inmolecular biology. This technique is used in biomedical research, criminalforensics, and molecular archaeology.Platingmethods In the laboratory various platingmethods are used for isolation, propagation and enumeration of microorganisms.All methods uses aseptic techniques in order to maintain sterile environment.Plating methods includes thefollowing; Streak-plate method Pour-plate method Spread-plate method Soft agar overlays method Replica-plate methodStreak plate method:It is used for isolation of pureculture of bacteria from mixed culture. In a colony millions of bacterial cellsIn this method the sample isspread over the agar plates with the help of sterile metal loop in such a waythat the bacteria can deposit over the plates separately and upon incubationthey develop into colonies.
Beforestreaking the loop is sterilized by passing it on a flame and is cooled beforepicking of inoculum. The streaking is performed in a zigzag manner. .
The plate contain more growth in the firstsection. The second section have less growth, while the final section possessleast amount of growth and the colonies were isolated.Pour platemethod:This method is used for determination ofmicroorganisms in a liquid sample. In this method 1ml of inoculum is poured inthe center of petri plate with the help of sterile pipette. 5ml of moltencooled agar is then poured into the plate containing the sample or inoculum andis mixed well. After solidification the plates were placed in the incubator for24-48 hours.Spread plateprocedure:This method is used for quantification ofbacteria in a solution. In this method a glass or metal spreader is used toapply small amount of suspended bacteria in a solution over a plate.
Like pourplate method serial dilutions are made for countable plate. 1ml of sample ispoured from the dilution series onto the agar plates. The sample is then spreadevenly with the sterilized L shaped spreader. The plates were then incubated at37C for 24-48 hours. CFU value of the sample is calculated.
After counting thecolonies multiply with the dilution factorSoft agaroverlays method:This is used for the detection ofbacteriophages that range in size from 100-200 nm. Phages require bacterialcells as a host for replication, therefor their propagation require mixture ofhost cells and phages. In this method 50 ?l to 200 ?l, of a phage suspension isplaced in a tube containing about 108 bacteria which is then spread on moltennutrient agar. Then this mixture is placed on the surface of hard nutrient agarplate. The plates were placed in incubator and after some time cloudyappearance of plate indicates growth of bacteria. Formation of plaquesindicates that phages are present and they infect the bacteria and cause theirlysis.Replica platemethod:This method allows the comparison betweenprimary and secondary plates.
This is useful for phenotype selection. Firstprimary plate is made by spread plate method. Secondary plate contain growthinhibitors or it lack a particular nutrient. Secondary plate is inoculated withthe cells from primary one. A piece of velvet is pressed on a primary plate sothat the bacterial cells adhere to it and is then transferred to varioussecondary plates. This allows screening of phenotypes in a single experiment.