Resistance Selection Method: This method isolates mutants that are resistant to antibiotics or bacteriophages by growing bacteria in their presence and selecting survivors. Wild type cells are generally not resistant. Replica plating allows screening colonies on different plates to identify phenotypes like antibiotic resistance. It creates copies of a master plate using velvet to inoculate colonies onto other plates.

5. Resistance Selection Method:
It is the other approach for isolation of mutants. Generally the wild type cells are not
resistant either to antibiotics or bacteriophages. Therefore, it is possible to grow the
bacterium in the presence of the agent (antibiotics or bacteriophage and look for survivors.
This method is applied for isolation of mutants resistant to any chemical compounds that
can be amended in agar, phage resistant mutants.

7. Replica plating is the technique by which each colony/clone is inoculated onto another plate
according to a numbered scheme. This method allows each clone to be tested by a variety of
methods, while retaining a master plate from which clones can be picked.
Principle
Replica plating involves creation of exact copy of master plate. The exact copy of master
plate can be created with the help of sterile velvet leather, cotton or chamois leather stamp
or clothes. These clothes or stamp acts as microneedle for inoculation of microbes when
pressed against master plate bacterial culture.
The purpose of replica plating is to be able to compare the master plate and any secondary
plates to screen for a selectable phenotype. For example, a colony which appeared on the
master plate but failed to appear at the same location on a secondary plate shows that the
colony was sensitive to a substance on that particular secondary plate. Common screenable
phenotypes include auxotrophy and antibiotic resistance.
Application of Replica plating
• Replica plating is a simpler method for screening of isolates that do not have
the ability to grow under specialized conditions.
• Classification of colonies differing from each other in a number of known
nutritional requirements, isolation of new mutant strains.

8. Procedure
• Mount a piece of sterile velvet by stretching it on a cylindrical metallic block
(slightly smaller than Petri dish).
• Place the block with velvet side facing upwards.
• Invert the Petri dish with the lawn of bacterial cells (master plate) and gently
press against the velvet. The number of projecting fibres of the velvet (almost
1000/sq. inch) act as inoculating needles sampling every clone of the cells in the
lawn.
• Remove the Petri dish and press two or more phage coated agar against the
velvet in turn.
• Save the original master plate.
• Incubate the subsequent phage coated plates.
• A few colonies appear on the phage coated plates. Some of these may
represent mutants that arose during the cell divisions that occurred after replica
plating.
• Colonies found at the identical positions on every replica plate can be
presumed to have arisen from an inoculum of phage resistant ones transferred
via the velvet from the phage resistant clone on the master plate

10. Resistant Selection Method
It is the other approach for isolation of mutants. Generally the
wild type cells are not resistant either to antibiotics or
bacteriophages. Therefore, it is possible to grow the bacterium
in the presence of the agent (antibiotics or bacteriophage and
look for survivors. This method is applied for isolation of
mutants resistant to any chemical compounds that can be
amended in agar, phage resistant mutants.

11. Substrate Utilization Method:
This method is employed in the selection of bacteria. Several bacteria utilize
only a few primary carbon sources. The cultures are plated onto medium
containing an 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 colour indicator
plates. A popular medium (EMB agar) is used for this purpose. The EMB agar
contains two dyes eosin and methylene blue in the medium. Colour of these
dyes is sensitive to pH. This medium also contains lactose sugar as carbon
source and complete mixture of amino acids.
Therefore, both lactose wild type (Lac+) and lactose mutant (Lac–) cells can
grow and form colonies on EMB agar plates. The Lac+ cells catabolize lactose
and secrete acids, therefore, local pH of the medium decreases. This results
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, possibly
ammonia is produced that increases the local pH and decolorizes the dye
resulting in white colony.

12. AMES /Carcinogenicity Test