Detection and Isolation of Mutants By: Replica-Plating Technique

Detection and Isolation of mutants by
Replica-plating technique
HISTORY
• Replica-plating technique was developed by
Esther and Joshua Lederberg in 1952.
• The experiment they performed helped to show

that many mutations are random, not directed.
• In the experiment they introduced colonies from

an original plate to new plates by "stamping" the
original plate with a cloth and then stamping empty
plates with the same cloth.
REPLICA PLATE METHOD
• It is a microbiological technique which we use to get one or more secondary
(replica) petri plates inoculated with the same colonies of microorganisms from
a primary (master) plate. The technique is so called, because each secondary
plate bears an exact replication of the colonies on the master plate.
Original
colonies Replica /
Secondary
plates
Replication of
colonies
Importance of replica plate technique
1. Detection of common observable phenotypes of microorganisms

2. Negative selection
3. Confirmation of the auxotrophic nature of microorganisms
4. Identification of the deficient biochemicals
5. Transferring colonies as genetic identical
The Procedure
Step 1
• The primary plate is prepared with distinct colonies. This is called the master
plate. A single master plate can be used to produce several replica plates.
or
Master plate
Step 2
• A sterilized block of wood (or cork) of a size suitable for the master plate is
covered with velvet cloth.
• Hereby, centre the velvet over top of the block and place the ring clamp with the
orientation mark facing up.
Block Wooden block Ring clamp Fixing the velvet

with the mark with the clamp
Rubber band
Step 3
• Place the master plate upside down lining the orientation mark with that of the
ring clamp’s.
• Let the colonies touch the velvet and gently come across the middle with just
fingertip pressure and then go around the edge.
Step 4
• Lift the master plate straight up and replace it on its lid.
Step 5
• Place a new plate without colonies upside down lining the orientation mark
with that of the ring clamp’s.
• Push down on the plate applying a light pressure with fingertips.
• Gently remove the plate by lifting straight. The same procedure can be followed
to get 5 to 6 replica plates. The newly inoculated plates are called as replica
plates.
An alternative pathway
• The procedure can also be carried out vice versa.
• That means to say, the sterilized wooden block covered with the velvet can be
lowered into the master plate till the velvet touches all the colonies.
• Then the block is withdrawn and gently lowered into a plate containing
selection medium so that the colonies get transferred to the secondary plate.
Tips
• It is better for the ring clamp, the master plate and the replica plates to have
orientation marks. So that the replication of any colony can easily be identified.
Orientation marks
Ring clamp Master plate Replica plate

• The procedure should be followed under sterile conditions.
1) Let the Bunsen burner give its flame throughout the procedure.
2) Sterilize the velvet inside a plastic bag using the autoclave.
3) Hold the velvet only by its corners and not anywhere in its middle.
4) Ensure enough distance between the top of the velvet and ring clamp so that
when the plate is placed on the top it would get enough space to settle.
The gap
5) When removing plates lift them straight up to avoid smudging.
Upward lifting
Master plate Replica plate

6) When done, lift the ring clamp straight up and off the Bunsen burner and
place the velvet flat contaminated side up, in a bin for autoclaving.
Upward lifting
Ring clamp Bunsen burner Velvet

DETECTION AND ISOLATION OF MUTANTS
Selective medium
• For the detection and isolation of mutants, replica plates containing selective
media should be used.
• A suitably harvested selective media allows only the desired mutant cells to grow
and produce colonies.
Minimal medium
• A culture medium for microorganisms that contains the minimal necessities for
growth of the wild-type is called a minimal medium. It contains only inorganic
salts, a carbon source, and water.
Auxotrophic mutants
• Auxo-trophy is the inability of an organism to synthesize a particular organic

compound required for its growth.
• Auxotrophic mutant is a mutant with a nutritional requirement not present in

the wild type organism.
Detection
• The colonies that develop on the selection medium plate are due to
wild type cells.
• In contrast, those colonies of master plate that fail to grow on the
minimal medium are auxotrophic mutants.
Isolation
• The mutant colonies are isolated using the replica plate technique from the
master plate and used for further investigations.
Ex:
- confirmation of their auxotrophic nature
Identification
- identification of the deficient biochemical of mutant
colony
Isolation of the
mutant colony
• The same approach can be used for detection of other types of mutants.
- antibiotic resistance
THE END
-SAHAN

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Detection and Isolation of mutants by

• The experiment they performed helped to show

• In the experiment they introduced colonies from

1. Detection of common observable phenotypes of microorganisms

Block Wooden block Ring clamp Fixing the velvet

Ring clamp Master plate Replica plate

Master plate Replica plate

Ring clamp Bunsen burner Velvet

• Auxo-trophy is the inability of an organism to synthesize a particular organic

• Auxotrophic mutant is a mutant with a nutritional requirement not present in

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