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 -
One level assay with standard curve -
Prepare five different dilutions S1, S2, S3, S4 and S5 of reference standard in the ratio of 4:5 or 1:1.25. Prepare U3 as the final dilution of unknown substance equal to the median level of the standard S3.
For preparing standard curve use 12 petri dishes to accommodate 72 cylinders or cavities. A set of 3 plates (18 cylinders or cavities) is used for each dilution. Drop six assay cylinders on the inoculated surface from a height of 12 mm, using a mechanical guide or other device to insure even spacing on a radius of 2.8 cm and cover the plates to avoid contamination. (Use of 100 mm diameter disposable type of plates are preferred.)
On each of three plates fill alternate cylinders or cavities with solution S3 and remaining 9 cylinders or cavities with one of the 4 dilutions of the standard solution. Repeat the procedure for other 3 dilutions of the standard solution. For each unknown preparation use a set of 3 plates (18 cylinders or cavities) and fill alternate cylinders or cavities with the sample solution and each of the remaining 9 cylinders or cavities with solution S3.
Incubate the plates for about 18 hours at the specified temperature and measure the diameters or the zones of inhibition. Calculate the results and potency of the unknown substance.
Two level factorial assay -
Prepare parallel dilutions containing 2 levels of both the standard (S1 and S2) and Unknown (U1 and U2). On each of four plates fill each of its four cylinders or cavities with a different test dilution alternating standard and unknown. Incubate the plates for about 18 hours at the specified temperature and measure the diameters or the zone of inhibition. Calculate the results and potency of the unknown substance.
Cork borer
Cork borer is sterilized and bored into agar surface to bore holes / wells. But Cork borer after repeat use looses its shape there by giving oblong holes and resulting into oblong zones. Oblong zones may lead into incorrect results. The results thus obtained lead towards approximation rather than perfection.
Deformity of cork borer mainly occurs during sterilization. Sterilization is done by means of alcohol flaming or by autoclaving. Circular part of cork borer is always sharp and thin to cut the agar into circular shape.
Although cork borer is easy to handle, it is less in precision due to deformed shapes. Instead of cylinders holes of 5 to 8 mm in diameter with the help of sterile cork borer may be bored in the medium or paper discs of suitable quality of papers may be used.(See annexure 1,2 and 3)
Cylinders
Assay cylinders are made of glass, porcelain, aluminium or stainless steel with outside diameter 8 mm ?0.1 mm, inside diameter 6 mm ±0.1 mm and length 10 mm ±0.1 mm. These cylinders are easy to clean, handle and are autoclavable. (See annexure 4 and 5)
  Role of Cylinders -
Cylinders form firm well on agar surface when dropped through cylinder dispenser and hold the solution in its place. The diffusion of assay substance takes place resulting in circular zone of inhibition for antibiotic assay and zone of exhibition for vitamin assay. Cylinders are machine made hence all piece are uniform and zones obtained are very circular unlike use of cork borer.
While placing cylinders manually on agar surface there are chances that cylinders may get tilted resulting into double marks on the agar surface.
These double marks may interfere in the resolution and clarity of zones.
Requirements as per Pharamacopeaia are that Cylinder assay gives precise results but placing of cylinders manually on agar surface is a great task. Accurate placing of cylinders can be achieved by using some kind of cylinder dispenser. Cylinder does not have sharp edges which on repeat use can turn into deformed shape. Cylinders on dispensing on agar do not get immersed, but remain firm on the surface.
Cylinder assay though mentioned in all the pharmacopeias is not popular in the pharmaceutical industry due to unawareness of assay accuracy of cylinder assay and non availability of cylinder dispenser and cylinders in the market. If at all cylinders are used, they are dispensed manually. Manual dispensing of cylinders on agar surface is tedious, haphazard and time consuming. They can rarely be placed at equidistance.
The reference of cylinder assay is official in all pharmacopeias (IP, USP, BP,EP and JP).
 
  Procedure Using Cylinder Plate Method -
Shaw dispenser mentioned in the internet is not easily available and the article shows that it is for dispensing bottle caps and not cylinders of bioassay.
A two level factorial assay or one level assay is based on diffusion of an antibiotic solution through the agar layer. Study individual test method requirements before carrying out bioassay of an antibiotic or vitamin. Refer Table 1.
Table 1
Parameter
One Level Assay
Two level assay
 
Cylinders
 
Six cylinders are required. They should be dropped from a height of 12 mm.
Besides cylinders should be placed at a distance of 28 mm from each other and at definite distance from center to avoid merging of zones.
 
Four cylinders are required. They should be dropped from a height of 12 mm.
Besides cylinders should be placed at a distance of 28 mm from each other and at definite distance from center to avoid merging of zones.
 
To place cylinders perfectly cylinder dispenser is used.
A comparison of Advantages of Innovated Cylinder Dispenser over Manually Placing of cylinders are given in table 2. (See photographs)
Table 2
Sr.
Cylinder dispenser
Manually placing of Cylinders
 
1.
Cylinders are dispensed accurately and meets pharmacopoeia requirements.
  
Manually cylinders are placed on agar surface. It is not a dispensing.
2.
Cylinders fall at the same rate on agar surface. Does not vary from cylinder to cylinder, plate to plate within analyst and person to person.
Cylinders do not fall at the same rate on agar surface and varies from cylinder to cylinder, plate to plate within analyst and analyst to analyst.
3. Cylinders are dispensed equidistantly from each other and from the center.
Cylinders cannot be dispensed equidistantly from each other and from the center.
4. Time saving. Time consuming procedure.
5. Job is easy and is done with accuracy. Skill is required to place cylinders accurately.
6.
No double marks are observed. Clarity of zones is sharp.
Frequency of getting more than one mark on each plate of agar surface is more. This affects the clarity of zones.
7. Precise results due to sharp zones. Imprecise results due to blur zones.
8. No repetition of assays. Repetitions may occur due to zone clarity.
 
  Salient Features of Cylinder Dispenser -
1. The material of construction of CYLINDER DISPENSER is Pharma grade stainless steel.
2. The dispenser is reusable.
3. It is autoclavable and can be sterilized by steam sterilization and by dry heat sterilization.
4. Autoclaving will not alter bioassay results.
5. Unique device for cylinder plate method in bioassay.
6. Compliance to Compendia requirements.
7. Tailor made, Available in Two different models to meet Pharmaceutical requirements.
8. Saves time and yields equidistant placement on agar surface.
9. Saves man power in placing cylinders equidistantly on agar surface.
10. Reliable, reproducible and precise results.
11. Economic.
12. Portable.
13. Reusable.
14. Elimination of risk of repeat analysis.
15. Eco friendly
Patented Product
 
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