Basic concepts are explained.

1. EXPERIMENT NO: 02
AIM : To estimate unknown concentration of
acetylcholine solution by interpolation bioassay
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 Interpolation method of bioassay is less time consuming and
yet reliable as compared to matching type of bioassay.
o Advantages: sensitivity of the tissue is first determined by
prior plotting a concentration response curve with a known
sample of acetylcholine.
o If the linearity of the curve is good then one can do very
accurate estimation of test substance as explained in the
procedure.

4. PROCEDURE:
Procedure for tissue mounting:
1. The fresh entire gastro-intestinal tract of healthy hen was
obtained and kept in 200 ml Tyrode solution.
2. Aeration was provided immediately to the tissue.
3. Ileum was cut at the identified point & was transferred to a
dish containing tyrode solution.
4. The 2cm long portion was cut & intestinal content was
removed & freed from other tissues.
5. A thread was tied at each end taking care that the lumen was
left open & thread does not cross it.

5. 6. The tissue was maintained for stabilization for 30 min with
washing for every 10 min.
7. Basal tension was adjusted to 0.5 gm, magnification was kept
5-7 folds & bath volume to about 15 ml.
8. With the proper care the tissue was mounted in an oxygen
delivered organ bath containing tyrode solution &
temperature was maintained at 37 + 2 0 C.
https://www.youtube.com/watch?v=4jUw6mkYoTU

6. Procedure for Interpolation Bioassay:-
1. Dose response of AcetylCholine was taken with the contact time of
90sec.
2. Repeat the first response till the height of the response obtained on the
graph is same to ensure the stability of tissue preparation
3. Take at least 4 responses with increasing dose of acetylcholine or till
you get the maximum response with higher concentration.
4. Take two responses for sub maximum dose and then select unknown
dose of drug which fall in linear portion of concentration response
curve
5. Properly labeled the graph.
6. Measure the height of response in mm and plot the log dose vs %
response curve.

7. Observations:

8. Observation table:

9. Calculation and result:

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

11. Thank You