Download (6Mb) - Etheses - Saurashtra University

Chapter-5
Synthesis of highly substituted pyrroles
5.7 Antimicrobial sensitivity testing
WELL DIFFUSION / AGAR CUP METHOD (Lt. General Raghunath D. 1998,
Ashok Rattan, 1998; Patel R., Patel K. 2004,)
In vitro effectivity of antimicrobial agents can be demonstrated by observing
their capacity to inhibit bacterial growth on suitable media. The production of a zone
depends on two factors namely bacterial growth and concentration of antimicrobial
agent. The hole/well punch method was first used by Bennett. This diffusion method
has proved more effective then many other methods. According to Lt. General
Raghunath the well technique is 5-6 times more sensitive then using disk method.
Principle
When antimicrobial substance is added in agar cup (made in a medium
previously inoculated with test organism) the redial diffusion of an antimicrobial
agent through the agar, produces a concentration gradient. The test organism is
inhibited at the minimum inhibitory concentration (MIC), giving rise to a clear zone
of inhibition.
Requirements
1. Young broth culture of a standard test organism
2. Sterile Mueller Hinton Agar plate
3. Solution of antimicrobial substance
4. Cup borer
5. Alcohol etc.
Inoculum preparation
Inoculum was prepared by selecting 4-5 colonies from slope of stock culture
of the indicator organism and emulsifying them in a suitable broth. The inoculated
broth was incubated at 37ºC till it equals turbidity of a 0.5 McFarland standard. This
happens in 2-8 h.
Department of Chemistry, Saurashtra University, Rajkot-360005 173