For educational purpose only, prepared as per Pharmacy council of India syllabus.

1. EXPERIMENT NO: 05
AIM : To estimate unknown concentration of
acetylcholine solution by three point 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:-
1. Three points bioassay involves two doses of the standard and one
dose of test solution.
2. Where two responses of standard drug and one response due to test
sample are taken into consideration.
3. The test response should be intermediate between the two responses
due to standard drugs.
4. Dose ratio for two doses of standard should be 1:2.
5. The calculation of the test solution is done by using the following
formula.

4. Procedure:
1. Isolation of Hens ileum:-Same as experiment no. 1
2. The DRC of standard & test samples is first obtained from the
responses due to graded doses.
3. From the DRC of standard, two standard doses are selected in
such a way that they have produced 25% & 50% of the
maximal response respectively & are designated
as S1 & S2
1. The test response should be intermediate between the two
responses due to standard drugs.
5. The responses of selected doses of standards & test are repeated
by using a latin square method.

5. Procedure contd:
6. Consider S1=A, S2=B, T=C & randomize the sequence of
recording responses due to respective dose by latin square
technique.
7. Record 3 sets of responses as ABC & BCA, CAB and than
labeled & fix the tracing.
8. Unknown concentration of the test solution is calculated by
using the formula given.

6. Advantage: Reduced error, reduced variability.
Disadvantage: Lengthy, Large amount of test
sample required.

7. Observation:

8. Calculations:

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