Introduction
The activity (potency) of an antibiotic or vitamin may be demonstrated under suitable conditions by their inhibitory or growth response effect on microorganism. To carry out bioassay of an antibiotic or vitamin, pharmacopoeia recommends turbidmetric assay or cup plate assay-
(References Current pharmacopeia- 1) USP, 2) EP, 3) BP 4) IP
Turbidmetric assay method – Refer pharmacopeia
Cylinder plate (Cup plate) assay method -
Refer to pharmacopeia for preparation of stock solution of standard. Further dilutions to be prepared using buffer solution or diluents as directed in the pharmacopeia. Apply the solutions to the surface of solid medium in sterile cylinders or in cavities prepared in the agar. The volume of solution added to each cylinder or cavity should be uniform and sufficient enough to fill the holes i.e. cylinders or cavities.
Cavities are created on agar surface with the help of cork borer or using cylinders. In cylinder plate assay the essential comparisons are restricted to relationships between zone diameter measurements within plates exclusive of the variation between plates in their preparation and subsequent handling.
A previously liquefied medium appropriate to the antibiotic (assay substance) and test organism (Refer pharmacopeia) should be poured into the petri dishes or large rectangular plates to give a depth of 3 to 4 mm except for Nystatin (1 to 2mm). Ensure uniform thickness of medium by placing plates on leveled surface.
Unless the base layer (without test organism) is solidified seed layer is not poured. Only in Nystatin Assay, base layer is not added. Direct seed layer is added. Incubate the plates for about 18 hours as indicated in the pharmacopeia. Accurately measure the diameters of zone of inhibition and calculate the results. Paper disc assay has certain limitations and is not widely used where as cork borer is used widely. There are two ways in carrying out cup plate assay method