SCREENING METHODS: Agar cup method

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The principle of the agar cup or agar well diffusion is the same as that of the agar disk diffusion method. A standardized inoculum culture is spread evenly on the surface of gelled gar plates. Wells of between 6 and 8 mm are aseptically punched on the agar using a sterile cork borer allowing at least 30 mm between adjacent wells and the Petri dish. Fixed volumes of the plant extract are then introduced into the wells (plate-2). The plates are then incubated at 37oC for 24 h for bacteria (Mbata et al., 2006, Panda et al., 2011b,d,e).

Agar dilution methods

The agar dilution methods are relatively quick methods that do not involve the use of sophisticated equipment. Any laboratory with basic microbiological facility can use this method. In this method, the test substance is incorporated at known concentrations into the agar. Once the agar set, bacteria are applied to its surface. Replicate dishes can be set up with a range of concentrations of the test substance and by dividing the surface of the agar into wedges or squares. A number of bacterial species may be applied to a single dish. In this way, a large number of bacteria may be screened within a single assay run. The dishes are incubated for 24 h or more and the growth of the bacteria on the extract/agar mix is scored either as present/absent or a proportion of the control (e.g. 0, 25%, 50%, 75%, 100%).
plate2Screening Methods in the Study-5
Plate 2: Photograph of agar diffusion method of various plants extract collected from Similipal biosphere Reserve, Odisha, India (Source-Panda et al., 2012)
a. Zone of inhibition against V. cholerae
b. Zone of inhibition against S. typhi

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