Preparation of antibiotic disc

Aim

To prepare antibiotic discs economically and to determine  the resistance pattern of  antibiotics against common pathogens.

Principle

Antibiotics are  defined as  antimicrobial substances which inhibit the growth of pathogenic bacteria. It is widely used in treatment of many diseases. Penicillin was the first antibiotic, discovered by Alexander Fleming in1928. Antimicrobial susceptibility testing can be studied  by different methods such as disk diffusion method, dilution method, E test  method. The disk-diffusion agar methods assess the effectiveness of antibiotics on a specific microorganism. Each disc is a standard size and is impregnated with a standard volume and concentration of the antibiotic. Impregnated  disc with a known concentration of an antimicrobial compound is placed on Mueller-Hinton (MH) agar which is swabbed with test organism .After incubation, the absence of growth around the antibiotic disks indicates the respected organism is susceptible to that antibiotic where as  presence of growth around the antibiotic disk indicates the organism is resistant to that particular antibiotic

Materials required

Whatmann No:1 filter paper, Punching machine, MHA plates, Antibiotic powder, bacterial species

Preparation of the filter paper discs

First step includes, the punching of holes in Whatmann No:1 filter paper of approximately 6mm diameter

The discs  were sterilized in an autoclave.

The sterilized discs were stored in screw capped bottles.

Preparation of antibiotic stock solution

·         Standard antibiotic powders were obtained commercially.

·         Known weight of antibiotic powder was dissolved in sterile distilled water to obtain the stock solution.( 1 g of sodium ampicillin  was dissolved in sterile distilled water and the volume was made upto 10 ml to get a concentration of 100 mg/ml. If sterilization is required, prewash the filter with  sterile distilled water. Then pass the ampicillin solution through the washed filter. Store the ampicillin in aliquots at -20°C for a year

Impregnation on the discs

Sterile discs were spread out in petri dishes approximately 5mm apart.

Using a micropipette, a fixed volume of 20μl was loaded on each disc.

The disc were allowed to dry and then stored in container in the refrigerator.

  Determination of  resistance pattern 

The fresh  broth of different isolates  were prepared and matched with 0.5 McFarland solutions.

The inoculum were then swabbed on prepared  Muller Hinton Agar plates

These plates allowed to stand at room temperature for 10 minutes(Pre diffusion time)

Antibiotic discs were placed  at equal distance to avoid overlapping of inhibition zone and at least 24mm away from edge of plate  

The plates were then incubated at 37oC overnight.

The zone of inhibition for each antibiotic was noted and the values were compared with the standard sensitivity values.

Depending on the values, the organism was classified as sensitive or resistant.

Resultl

The diameter of the zone of inhibition was measured at the end of incubation period. The zone diameter was recorded to the nearest millimeter…..mg of ampicillin was given….mm zone of inhibition against ………sp


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