If we work with only top agar and prepared well in it and add antibiotic...Do we get result? or it is necessary to add first base agar and later top agar with bacterial culture?
I understand, your purpose is to perform an antimicrobial susceptibility assay.
I suggest to go for a broth dilution assay, that will be easy to handle, and you will be able to perform the assay with lesser amount of antibiotic.
ADDITION OF BASE AGAR IS TO HAVE GET CONTRAST OF ZONE OF INHIBITION OR CLEARANCE
The original method of bioassay was developed for penicillin (of course because it was the first antibiotic). Research on penicillin was pioneered at Oxford University and so here the first method was designed. This was done by Abraham and Chain in 1941.
This original method didn't used two layers of agar.
However many modifications of Abraham's Cylinder Plate method were proposed later. Most significant work was by SCHMIDT and MOYER. They presumably were the first to to suggest use of two layers in 1943.
There are plenty of reasons to use two layers.
1. They used different media for supporting growth (base) and to have sharp boundaries of zone of inhibition (top agar with more salts).
2. On those days workers used to prepare large number of plates and it was common practice in their laboratory to prepare base agar 24 hrs earlier (probably to have uniformly dried plates) and add top agar for assay on next day.
3. They were aware of many factors that influence size of the zone of inhibition and thus final result. One of these factors is depth of agar that increases/decreases diffusion of antibiotic solution and changing the diameter of zone. In order to standardise this they had a regular practice of having 22 ml of base agar prepared early overlaid with 3 ml of top agar on the day of assay.
Original paper can be found on following link, a strongly recommended reading. :)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373892/pdf/jbacter00690-0091.pdf
Thank you Vijay Kothari, Mukesh Pimpliskar and Jay Bergi..Paper is really good one.
@Dr. Mukesh Pimpliskar
Don't we see a zone of inhibition without using basal agar? Cause many of the antibiotic sensitivity tests use a single layer of agar and still get a crisp zone of clearance.
I use Muller Hinton agar (single layer) and spread 200ul of inoculum (OD 0.5) to get clear zones of inhibition.
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