AIM: Assay of antibiotic resistance by disc diffusion method

Requirement

24 hours old culture

Antibiotic disc of various kind ( Penicillin, Erythromycin, Streptomycin, Ciprofloxacin, Cefixin)
Ethanol
Sterile cotton swabs or spreader
Alcohol
Ruler
Bunsen burner
Inoculum
innoculating loop
Forceps
Petriplate
Marker
prepared saline
Laminar air flow

Reagent preparation;
for 1000 ml saline preparation
1000ml = 58.5 gm of Nacl
so, for 100ml = 58.5/1000 100
5.85gm Nacl
5.85 gm of Nacl in 100ml distilled water is added to make 100 ml of saline

Inoculum preparation
 1. using a sterile inoculating loop or noodles touch four or five isolated colonies of the
organism to be tested

2. suspend the organism in 2ml of sterile saline

3. vortex the saline tube to create a smooth suspension

4. use this suspension within 15 min of preparation

Principal

A pure bacterial culture is suspended in saline, its turbidity is standardized, and it is swabbed
uniformly across an agar plate. An antibiotic- or extract -impregnated filter paper disk is then
placed on the surface of the agar. The concentration of these constituents will be highest next to
the disk and will decrease as the distance from the disk increases. If the antibiotic or extract is
effective against bacteria at a certain concentration, no colonies will grow where the
concentration in the agar is greater than or equal to the effective concentration. This is the zone
of inhibition. In general, larger zones of inhibition correlate with lower minimum inhibitory
concentration (MIC) of antibiotic or extract for that bacterial strain. An exception to this is when
molecules of the antibiotic or extract are larger or hydrophobic because these diffuse through the
agar slowly.

Protocol

1.Prepare the nutrient agar media and autoclave the media sline and glasswares.

2.Pour culture media into a sterile petriplate

3.Inoculate the entire agar surface of each plate, first in horizontal direction and then in vertical
direction to ensure the even distribution of the bacteria over the agar surface, using the swab.

4.Allow the agar surface dry for 5 minutes.

5. sterilize the forceps with alcohol before picking up antibiotic disc.

6.We have used different concentration of antibiotics and placed them into different area.
7. Sterile forceps were used to press the disc in place was marked by antibiotic name.

8.Incubate the plate upside down for 24 hours at 37degree celsius.

Observations and Results

1. Examine all the plates for the zone of inhibition surrounding the discs.
2. Measure diameter of zone of inhibition in mm, using the ruler on the underside of the plate.
3. Record the zone size and prepare a graph comparing the zones obtained with the known
concentrations versus the zone of unknown.
4. Find out the approximate number of penicillin units in the culture filtrate.

Antibiotics Zone of inhibition Sensitive/Resistance

Penicillin Resistance
Erythromycin 3cm Sensitive
Streptomycin Resistance
Cefixin 4cm Sensitive
Ciprofloacin 4.5cm Sensitive

Fig - 1 Fig-2
Fig-3 Fig-4

Precautions

1. The surface should be moist but without droplet of moisture.

2. The antibiotic disks should be maintained at 8℃ or lower or freeze at -14℃ or below
until needed, according to the manufacturer’s recommendations.
3. Allow the disks to warm room temperature before use.
4. Don’t use expired disks.