ISOLATION OF MUTANTS

MADE BY:SHVETA ARYA

B.PHARM

ISOLATION OF MUTANTS:

Mutation occurring in microorganism can be

detected and efficiently isolated from the parent

organism of other mutants.

While studying we must be aware of wild type

characters of an organism ,so the mutants can

easily detected.

In bacteria and other haploid microorganism, the

detection system are straight forward because any

new allele should be observed immediately.

• In albino mutation, the detection is very simple. It

requires only change in colour of bacterial colony.

• The other detection systems are rather complex.

SOME DETECTION METHODS

Replica plating technique.

Resistance selection method.

Substrate utilization method.

Ames method

1. REPLICA PLATING

TECHNIQUE:

Joshua and Esther Ledgerberg (1952) developed a

new technique called replica plating .

This technique is used to detect auxotrophic

mutants and wild type strains on the basis of ability

to grow in the absence of amino acids.

Also this test is used to demonstrate the presence

of antibiotic resistance in bacterial cultures prior to

exposure of antibiotic

STEPS INVOLVED

Generate the mutants by treating a culture with a

mutagen e.g.nitrosoguanidine .

Inoculate a plate containing complete growth

medium and incubate it at proper temperature. Both

wild type and mutant survivors will from complete

medium.

This plate containing complete medium is called

master plate.

Prepare a piece of sterile velvet and gently on the

upper surface of the master plate to pick up

bacterial cell from each colony.

As pressed the master plate, again gently press

the velvet on the replica plates containing complete

medium in one set and lacking cine in only leucine

in the other set.

Thus, the bacterial cells are transferred in replica

plates in the same position as in master plate.

Incubate the plates and compare the replica plate

with master plate for bacterial colony not growing

on replica plate..

2.RESISTANCE SELECTION METHOD

This is another method used for isolation of

mutants.

Generally the wild the wild type cells not resistant

either to antibiotics or bacteriophage.

Therefore, it is possible to grow the bacterium in the

presence of agent.

This method is applied for isolation of mutants

resistant to chemical compounds that can be

amended in agar, phage resistant mutants.

3.SUBSTRATE UTILIZATION METHOD :

This method is employed in the selection of bacteria. Several bacteria utilize only a few carbon sources.

The cultures are plated on to medium containing alternate carbon sources.

Any colony that grows on medium can use the substrate and are possibly mutants. These can be isolated.

Sugar utilization mutants are also isolated by means of color indicator plates.

EMB medium is used for this purpose.

This medium contain lactose sugar as carbon source and complete mixture of amino acids.

Therefore both lactose wild type and lactose mutant

cells can grow and form colonies on EMB agar

plates.

The lac+ cells catabolize lactose and secrete

acids,therefore the pH of the medium decreases.

This will result in staining of colony to dark purple.

On the other hand, Lac- cells are unable to utilize

lactose and use some of the amino acids as carbon

source.

After utilization of amino acid, ammonia is produced

that increases the pH and de colorize the dye

resulting in white colony.

4. AMES TEST :

4. Ames test In 1974 Bruce Ames developed a

method for evaluating the potential of chemical to

cause cancer, known as Ames test .

Ames test is based on the principle that both

cancer and mutations results from the damage of

DNA, and results of experiments have

demonstrated that 90% of known carcinogen are

also mutagens.

Several species of salmonella typhimurium are

employed. Each strain contains a different mutation

in the operon histidine biosynthesis.

STEPS OF AMES TEST:-

Prepare the culture of Salmonella histidine

auxotrophs (His-).

Mix the bacterial cells and test substance(

mutagen) in dilute molten top agar with a small

amount of histidine in one set, and control with

cmplete medium plus large amount of histidine .

Pour the molten mix on the top of minimal agar

plates and incubate at 37°C for 2-3 days.

Until histidine is depleted all the His- cells will grow

in the presence of test mutagen.

When the histidine is completely exhausted only

the revertants will grow on the plate.

The number of spontaneous revertants is low,

whereas the number of revertant induced by

carcinogen is quite high.

High number of colonies represent the greater

mutagenicity.

o A mammalian liver extract is added to the above

molten top agar before plating.

o The extract converts the carcinogen in to

electrophilic derivatives which will soon react with

DNA molecule.

o In natural way it is occurs in mammalian system

when foreign particle are metabolized in the liver.

o Bacteria does not have metabolizing capacity,

therefore, the liver extract is added to this test, to

promote transformation.

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