This document describes an experiment to estimate the unknown concentration of an acetylcholine solution using a bracketing bioassay with hen's ileum tissue. The experiment involves determining the dose-response curve for a standard acetylcholine solution and then bracketing responses to the unknown solution with responses to the standard. Close bracketing of the unknown response between two standard responses allows the concentration of the unknown to be accurately calculated using the formula provided. The aim is to present the methodology for using a bracketing bioassay to estimate an unknown drug concentration.

1. EXPERIMENT NO: 03
AIM : To estimate unknown concentration of
acetylcholine solution by bracketing bioassays
using hen’s ileum.
Presented by:
Prof. Mirza Anwar Baig
Dept of Pharmcaology
AIKTC, School of Pharmacy, New Panvel

2. REQUIREMENTS:
1. Basal tension – 0.5 gm
2. Extra load- 1 gm
3. Animal – Hen’s ileum tissue
4. Drug – Acetylcholine stock solution (10 μg/ml) & test solution.
5. Physiological solution – Tyrode solution
6. Contact time – 90sec
7. Magnification – 5-7 times.

3. PRINCIPLE:-
o Matching & bracketing method is very useful if the amount of
material is small and if the sensitivity of the tissue is not
steady.
o The results of the assay do not depend on the actual
measurement of response.
o It is decided whether a particular concentration of test
solution is greater or smaller than that produced by the dose
of the standard

4. Bioassay by Bracketing Method
 Performed by selecting two standard doses, which will give a close
bracket on either side of the response produced by the unknown.
 The working dose of standard is first determined in the sensitive part
of dose-response curve, that is, a dose that will approximately
produce 50% of the maximal concentration.
 The dose of the standard drug is kept constant throughout the
experiment, in order to have some idea about the change in the
sensitivity of tissue with time.
 The standard drug is added at fixed intervals but alternating with the
test so that each response produced by a dose of test substance is
bracketed by responses produced by the dose of standard.
 Close bracketing gives more accurate results.

5. Observation on kymograph paper:

6. Formula to calculate concentration of unknown drug:

7. Calculations:

8. Result:

9. References:
1. Kulkarni S. K. Handbook of Experimental
Pharmacology, Vallabh Prakashan, New Delhi.
2. Ghosh M.N. Fundamentals of Experimental
Pharmacology Hilton & Company, Kolkata

10. Thank You