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Guru Nanak Institute of Pharmaceutical Sciences and Technology

Subject: Genetic Engineering Lab Subject Code: MSBT/MSMC/MSGN-291

SL NO. EXPERIMENT NO. DATE:

AIM: ISOLATION OF ANTIBIOTIC-RESISTANT MICROORGANISM BY


EXPOSING AN AGAR PLATE CONTAINING CONCENTRATION OF GRADIENT OF
ANTIBIOTIC TO AN INOCULATION OF MICROORGANISM TO BE TESTED.

Objective:

 How to prepare gradient plate


 Isolation of antibiotic resistance microorganism by gradient plate method.

A. PRINCIPLE:

An excellent way to determine the microorganisms that are resistant to antibiotic is to grow them
on a gradient plate of a particular antibiotic. The gradient plate consist of two wedges like layers
of media, a bottom layer of plain nutrient agar and top layer of antibiotic with nutrient agar
medium. 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 particular antibiotic
in the medium to check the microorganism either resistant or not. The colonies develop in the
high concentration are resistant to the action antibiotic , and are considered as resistant
microorganism against that antibiotic.
B. MATERIALS REQUIRED:

 24 hour old nutrient broth culture of Escherichia coli.


 Two nutrient agar deep tubes ( 10 ml per tube)
 Amoxicillin and doxycycline solution ( 40 μg/ml & 80 μg/ml)
 A beaker with 90% ethanol
 Sterile petri plates
 Sterile 1 ml pipettes
 Glass rod spreader
 Water bath

C. PROCEDURE:

1) Preparation of gradient plate


 Melt two nutrient agar plates maintained at 96˚C and cool to 55˚C.
Guru Nanak Institute of Pharmaceutical Sciences and Technology
Subject: Genetic Engineering Lab Subject Code: MSBT/MSMC/MSGN-291

 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.
 After the agar medium is solidified remove the glass rod and place the
plate in the horizontal position.
 Pipette out 0.1 ml of amoxicillin and doxycycline solution of 40 μg/ml and
80 μg/ml into the second tube of the second nutrient agar medium.
 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.
 Label the low and high antibiotic concentration area on the bottom of the
plate.
2) Inoculation of culture:
 Pipette out 100 μl of the overnight Escherichia coli culture onto the
gradient plate after 24 hours of its preparation.
 Spread the inoculums evenly over the agar surface with a sterile bent glass
rod by rotating the plate.
 Incubate the inoculated plate in an inverted position at 37˚C for 24-48
hours.
 Observe the plate for appearance of Escherichia coli colonies in the area
of low antibiotic concentration and high antibiotic concentration and
record the results.

D. OBSERVATION AND RESULTS:

E. REMARKS :-

ATTENDANCE VIVA PERFORMANCE (20) TOTAL


(10) (10) (40)
MANUAL CONDUCT METHOD CLEANLINESS
MAINTENANCE & ACCURACY (5)
(5) INVOLVEMENT (5)
(5)

Signature of the Teacher