Bioassay of Serotonin on Rat Fundus Strip by Three Point Bioassay

Introduction

Bioassay is a quantitative method used to determine the concentration or potency of a biologically active substance by measuring its effect on living tissues. Serotonin (5-Hydroxytryptamine, 5-HT) is an important endogenous neurotransmitter that produces contraction of smooth muscles, particularly in the gastrointestinal tract.

The rat stomach fundus strip is highly sensitive to serotonin and produces reproducible graded contractions. Therefore, it is widely used for bioassay studies in pharmacology laboratories.

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Aim

To determine the concentration of an unknown sample of serotonin by three point bioassay method using isolated rat fundus strip.

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Principle

Serotonin produces graded contractions of the rat fundus strip by acting on specific serotonin receptors present in the smooth muscle. The height of contraction is directly proportional to the logarithm of the dose within a certain range.

In the three point bioassay method, two doses of standard drug (S1 and S2) and one dose of test drug (T) are selected. The test dose is adjusted so that its response lies between the responses of the two standard doses. By comparing the responses, the potency or concentration of the unknown sample can be calculated.

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Requirements

• Rat fundus strip

• Organ bath containing physiological salt solution (Tyrode solution)

• Aerator (oxygen supply)

• Isotonic transducer or simple lever setup

• Kymograph or data acquisition system

• Standard serotonin solution

• Test serotonin solution

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Procedure

1. Sacrifice the rat humanely according to ethical guidelines.

2. Isolate the stomach and carefully cut the fundus portion.

3. Prepare a longitudinal strip of fundus tissue.

4. Mount the strip in an organ bath containing oxygenated Tyrode solution maintained at 37°C.

5. Allow tissue to equilibrate for 30–45 minutes.

6. Apply increasing doses of standard serotonin to obtain graded responses.

7. Select two standard doses (S1 and S2) producing submaximal responses.

8. Apply the test dose (T) in between the two standard doses.

9. Record responses in the sequence S1 – T – S2 (repeated for accuracy).

10. Measure the height of contractions and calculate the unknown concentration.

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Observations

• Serotonin produces dose-dependent contraction of rat fundus.

• The response increases with increase in concentration.

• A linear relationship is observed in the middle portion of the dose-response curve.

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Advantages of Three Point Bioassay

• Simple and reliable method

• Requires fewer doses

• Accurate for estimating potency

• Suitable for student practicals

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Applications

• Determination of drug potency

• Study of drug-receptor interactions

• Standardization of biological preparations

• Pharmacological research and teaching

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Summary 

The three point bioassay of serotonin using rat fundus strip is a reliable and widely accepted method for determining the potency of serotonin. The experiment clearly demonstrates the concept of graded response, dose-response relationship, and quantitative evaluation of drugs in experimental pharmacology.

This practical builds strong foundational understanding of receptor pharmacology and bioassay techniques for pharmacy and medical students.

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