AIM: To become familiar with gradient plate method for isolating
antibiotic resistant mutants and to perform replica plating procedure for
isolating and identifying Auxotrophic mutants.
Principle
Mutations often inactivate a biosynthetic pathway of the microorganism, and frequently make a microorganism unable to grow on a medium lacking an adequate supply of the pathway’s end product. Based on this principle microorganism are classified as Prototrophic and Auxotrophic. Prototrophic organisms (wild type) have the same nutritional requirements as that of their ancestors. They need only inorganic salts, an organic energy source such as sugar, fat, protein and water to survive and grow. That is, the Prototroph’s need only "Minimal medium" for their growth and survival. Auxotrophic mutants are unable to grow without one or more essential nutrients. Auxotrophs are mutant for particular nutrient synthesis pathway enzymes. Such an error is known as an inborn error of metabolism, whether it occurs in a bacterium or a eukaryote. An auxotroph can be grown only on an enriched medium that provides the particular nutrient that the mutant cannot metabolize on its own.
Gradient Plate Technique:
The gradient plate
consists of two wedges like layers of media: a bottom layer of plain nutrient
agar and top layer of antibiotic with nutrient agar. The antibiotic in the top
layer, diffuse into the bottom layer producing a gradient of antibiotic
concentration from low to high. A gradient plate is made by using
Streptomycin in the medium. E. coli, which is normally sensitive to
Streptomycin, will be spread over the surface of the plate and incubated for 24
to 72 hours. After incubation colonies will appear on both the gradients. The
colonies develop in the high concentration are resistant to the action of
Streptomycin, and are considered as Streptomycin resistant mutants. For
isolation of antibiotic resistant of gram negative enteric bacteria, the
antibiotics commonly used are Rifampicin, Streptomycin, and Erythromycin etc.
Replica Plating Method:
The technique was developed by Joshua and
Esther Lederberg in 1952 for providing the direct evidence for the existence of
pre-existing mutations. This technique isolates both nutritional mutants and
antibiotic resistant mutants. Their actual experiment concerned with
replicating master plates of sensitive cells to two or more plates containing streptomycin.
Replica plating allows the observation of microbes under a series of growth
conditions. The bacteria are grown in an environment that is not selective for
given mutation. This technique is used to transfer the members of each
colony to a selective environment. A simple velveteen covered colony transfer
device is used to transfer the colonies in nutrient agar medium supplemented
with or without a particular antibiotic or nutrient. The fibers of velvet
act as fine inoculating needles, picking up the bacterial cells from the
surface of this master plate. The velvet with its attached microbes is then
touched to the surface of a sterile agar plate, inoculating it. In this manner,
microbes can be repeatedly stamped onto media of differing composition. By
comparing the presence of colonies following incubation we can indirectly
determine the mutant colonies by their absence in the selective environment. A
colony that develops on a complete medium fail to develop on a minimal medium
that lacks a specific growth factor, the occurrence of a nutritional mutant is
indicated. The microbes that do not grow on the minimal medium represent
auxotrophic strains. The microbes that do
not grow on the minimal medium represent auxotrophic strains.
Replica plating technique
Materials required: Gradient Plate
Technique
1.
24 hour old nutrient broth culture of Escherichia
coli.
2.
Two nutrient agar deep tubes (10 ml per tube/culture).
3.
1% Streptomycin sulphate solution (100 µg/ml).
4.
A beaker with 90% ethanol.
5.
Sterile Petri plates.
6.
Sterile 1 ml pipette.
7.
Glass rod spreader.
8.
Water bath.
Procedure: I) Preparation of gradient plate:
1.
Melt two nutrient agar plates maintained at 960C and
cool to 550 C
2.
Pour the contents of one agar tube into a sterile petriplate.
Allow the medium to solidify in a slanting position by placing either a glass
rod under one side.
3.
After the agar medium is solidified remove the glass rod and place
the plate in the horizontal position.
4.
Pipette out 0.1mL of 1% Streptomycin solution into the second tube
of the second nutrient agar medium.
5.
Rotate the tube between the palms and pour contents to cover the
gradient layer agar and allow to the medium to solidify on a level table.
6.
Label the low and high antibiotic concentration area on the bottom
of the plate.
II) Inoculation of culture:
1.
Pipette out 200µl (0.2ml) of the overnight Escherichia
coli culture onto the gradient plate after 24 hours of its
preparation.
2.
Spread the inoculums evenly over the agar surface With a sterile
bent glass rod by rotating the plate.
3.
Incubate the inoculated plate in an inverted position at 37o C
for 48-72 hours.
4.
Observe the plate for appearance of E.coli colonies in the area of
low streptomycin concentration (LSC) and high streptomycin concentration (HSC)
and record the results.
Results: Colonies which appear in the area
of high concentration streptomycin region will be streptomycin resistant
mutants.
Replica plating method:
Materials required:
1. 24 hour old nutrient broth culture of Escherichia coli.
2. Minimal salt agar with glucose.
3. Three 10ml Nutrient agar deeps.
4. 1% Streptomycin sulphate solution (10mg /100ml of sterile water).
5. Sterile petridishes.
6. Sterile velveteen colony carrier.
7. Glass rod or wooden dowel stick .
8. Beaker with 95% ethanol.
9. Bent glass rod.
10. Quebec colony counter
Procedure:
DAY 1
1.
Melt the nutrient agar deeps tubes in a hot water bath maintained
at 960 C.
2.
Allow the molten medium to cool to 550 C.
3.
Pour the molten agar medium to two sterile petriplates and allow
to solidify in a horizontal position.
4.
Add 0.1% of Streptomycin, using sterile pipette into the third
tube of molten nutrient agar (maintained at 550C ), properly mix by
rotating between the hands and pour the contents into a sterile petriplate.
Allow to solidify.
5.
Add 200 µL of(0.2mL) of the the E.coli test
culture to the surface of the nutrient agar plate.
6.
Using an alchohol dipped and flamed bent glass rod spread the
inoclum evenly on the plate.
7.
Incubate the plate in an inverted position for 24-48 hours at
37 0 C.
8.
After incubation observe the colonies of E.coli on
the plate and this plate was considered as the master plate.
DAY 2
1.
A reference mark (at 12 O'clock position) was noted on the bottom
of the master plate, plate with nutrient agar and plate supplemented with
Streptomycin.
2.
The sterile velveteen colony carrier was carefully lowered and
gently pressed onto the colonies of the E.coli on the master
plate.
3.
Without altering the position of the carrier, the sterile velveteen
was gently pressed on to the nutrient agar plate followed by the Nutrient agar
plate supplemented with Streptomycin.
4.
Incubate both the inoculated plates, nutrient agar and
streptomycin agar plates in an inverted position for 48-72 hours at 370C.
Master plate is refrigerated.
5.
Following incubation number of colonies in the replica plates with
nutrient agar and streptomycin agar was counted using Quebec Colony Counter.
6.
The colonies appearing on the nutrient agar plates and
Streptomycin plates were noted and compared.
Results:
E.coli colonies which appear on the Streptomycin supplemented
agar was confirmed as Streptomycin resistant mutants and the number of colonies
were counted using Quebec Colony Counter.
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