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 37^o 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.

III. Confirming presence of Streptomycin resistant colonies of E.coli.

 

1. Select and mark an isolated colony of E.coli in the HSC region of the Nutrient agar plate.
2. Pick the selected colony with a sterile inoculating loop and streak on to second gradient plate towards the HSC region.
3. Repeat this step with one or two colonies of streptomycin resistant mutants from the HSC region.
4. Incubate the inoculated plates in an inverted position at 37^o C for 24-72 hours.
5. Observe the growth of streaked colonies towards the HSC region.

Results:

Growth  of E.coli colonies in HSC area indicates the successful isolation of 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. fter 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 velvetteen 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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