This document discusses various methods for detecting mutations in microorganisms. It describes that mutations can be detected if they cause an altered phenotype. It then explains several techniques for detecting mutations including replica plating, resistance selection, substrate utilization, and the Ames test. Replica plating involves transferring bacterial colonies from a "master plate" to plates with different growth conditions to identify mutants unable to grow in certain conditions. Resistance selection and substrate utilization detect mutants able to grow in the presence of antibiotics or use alternative carbon sources. The Ames test uses Salmonella strains to identify whether chemicals cause mutations detectable as increased reversion to histidine prototrophy.

1. Submitted to :
Presented by-
.

2. 2

3. •Detectability of mutations is a function of
their effect on the phenotype
• The likelihood of a given mutation being
detected is a function of a variety of
factors,
• All of which reflect the fact that one
detects only those mutations that
display a sufficiently altered phenotype
• UV. An inspection of the genetic code
will show that the majority of these will
cause the substitution of one amino
acid for another
3

4. 4
 The mutations that cause such
amino acid changes are termed
missense mutations
 It is particularly common for
temperature conditional mutants
to have significant amounts of
activity under the non-permissive
condition.
 The following arguments will
suggest the ways in which a
partial loss of a product function
would affect the phenotype and
therefore the detectability of the
mutant.

5. 5
 Mutations induced due to
radiations or due to
chemicals could be
broadly of two types - (i)
lethal mutations,
 (ii) visible mutations. Both
these types could be
located either on sex
chromosomes or on
autosomes .

6.  his method involves use of a ClB stock which carries
 (i) an inversion in heterozygous state to work as
crossover suppressor (C),
 (ii) a recessive lethal (l) on X-chromosome in heterozygous state,
and
 (iii) a dominant marker, Barred (B)for the barred eye.
 One of the two X-chromosomes in a female fly carried all these three
features and the other X-chromosome was normal.
 Male flies irradiated for induction of mutations were crossed
to ClB females. Male progeny receiving ClB X-chromosome will die
6
20YY 20YY
GrowthShowsSales

7. 7

8. 8
 The methods described in the preceding section were meant for
detection of sex linked lethals.
 For detection of sex linked visible mutations, Muller-5 and
attached X-chromosomes were used. The attached X females
(XXY) have a special advantage.
 When these females are crossed to an irradiated male, X-
chromosome of irradiated male goes either to superfemale
daughters or to the sons.
 Since in sons there is a single X-chromosome, any visible
induced mutation will immediately express itself and can be
easily scored

9. 9

10.  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.
10

11.  Replica plating technique.
 Resistance selection method.
 Substrate utilization method.
 Ames method
11

12.  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
12

13.  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.
13

14.  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.
14

15. 15

16.  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.
16

17.  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. 17

18. 18
 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.

19.  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
19

20.  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.
20

21.  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.
21

22. 22

23.  . Microbial Genetics” by John Cronan and
David Freifelder
 “Microbial Genetics (Jones and Bartlett Series
in Biology)” by Stanley Maloy and John Cronan
23

24. 24