Objective Principle Requirements Experimental specifications (conditions) Preparation of serotonin stock solution Preparation of Krebs solution (PSS) Procedure Kymograph recording of contractions Observation table Calculation Result and interpretation

1. ExperimentNo. 1
Bioassay of serotonin using rat fundus strip
by three point bioassay.
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5

2. Overview of Discussion
• Objective
• Principle
• Requirements
• Experimental specifications (conditions)
• Preparation of serotonin stock solution
• Preparation of Krebs solution (PSS)
• Procedure
• Kymographrecording of contractions
• Observation table
• Calculation
• Result and interpretation
2

3. Objective
To determine unknown concentration of serotonin
(5- hydroxytryptamine, 5-HT) using rat fundus strip
by three point bioassay.
3

4. Threepoint bioassay
4
In such a type of multiple point bioassay, two responses of
standard drug (S1 and S2) and one response due to test
sample (T) are taken into consideration. The selection of
two responses of standard should be such that they lie on
the linear portion of the concentration response curve and
also the ratio between the doses should be 1:2. The test
response should be intermediate between two responses
of standard drug.
The precision, reliability and reproducibility of this bioassay are
very high. It is most commonly used for estimating the
concentration of the unknown sample.
Principle

5. 5
Rat fundus is very sensitive tissue for the study of the action
of several naturally occuring substances like 5-
hydroxytryptamine, histamine, ACh and bradykinin. Unlike
the intestinal smooth muscle (ileum), fundus preparation is
slow contracting and slow relaxing type of tissue. It is
generally employed for the bioassay of serotonin. The
fundus (the upper part of the stomach) is grey in colour and
therefore, easily identified from the pylorus (pink in colour).
A zig-zag preparation of fundus strip is prepared so as to
exposed maximum portion of the tissue to drug.
The tissue is sensitive to 1 ng/ml of serotonin, 0.05 ng/ml of
histamine and 0.2-0.5 ng/ml of ACh.

6. 6
Mechanism of contraction
Serotonin acts on 5HT2B- subtype of serotonergic receptor
which function through Gq protein and activate membrane
bound phospholipase C (PLc) → generating inositol
trisphosphate (IP3) and diacylglycerol (DAG), the secondary
messengers release Ca2+ intracellularly → cause
depolarization and thereby contraction of gastric fundus.
Overnight fasted rats are used to record better response of
serotonin on isolated rat gastric fundus .

7. Requirements
Animal: Albino rats (150-200 g, overnight fasted)
Physiological solution: Krebs solution.
Drug- Serotonin (Stock solution: 10 mcg/ml)
Chemical- Fixing solution.
Instruments: Sherrington recording drum , Student organ
bath, Aerator, Insulin or tuberculin syringe to inject drugs
in small fractions, Dissecting board and various dissecting
instruments. Frontal writing lever and stand, Pipette,
Stop watch etc.
Miscellaneous: Kymograph paper, plasticin, clips, and
thread.
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8. Experimentalspecifications(conditions)
 Isolated tissue- Isolated rat fundus strip preparation
 Drug- Serotonin (Stock solution: 10 mcg/ml)
 Physiological salt solution (PSS)- Krebs solution
 Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact
time- 90 seconds, Washing period- 3 minutes
 Applied load/ tension- 1 g
 Bath capacity- 40 ml
 Bath temperature- 37°C
 Speed of rotation of drum- 0.25 mm/ second
 Magnification value (Mf) = d (F-W)/ d (F-T)
 Aeration- Normal air (1- 2 bubbles/ second)
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9. Preparationof serotoninstock solution
Stock solution- 10 μg/ml
 Dissolve 1 mg of serotonin in 100 ml of distilled water → 10
μg/ml
9

10. Preparationof Krebs solution (PSS)
 Prepare 1 litre of Krebs solution by dissolving NaCl (6.9
g), KCl (0.35 g), MgSO4.7H2O (0.28 g), NaHCO3 (2.1 g),
KH2PO4 (0.16 g) and glucose (2.0 g) in distilled water.
 MgSO4.7H2O should be added at last.
 CaCl2 (0.28 g) should be dissolved separately in distilled
water to avoid chances of precipitation of salt.
 Mix CaCl2 solution to the higher volume of PSS.
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11. Procedure
 Sacrifice the rat by a blow on the head and carotid bleeding.
 Cut open the abdomen and expose the stomach.
 Identify the fundus of the stomach (upper greyish part). Incise it
from the junction of pyloric part (pink in colour) and put it in the
dish containing Krebs solution.
 Incise the fundus from the lesser curvature and open it
longitudinally. Give alternate zig-zag cuts to make a fundal strip
preparation. Tie both the ends with the thread and mount in the
organ bath containing Krebs solution at 37°C. Aerate the tissue.
 Apply 1 g load and allow the preparation to equilibrate for 30
min. Using frontal writing lever with 10-12 magnification, record
the contractions due to increase concentrations of serotonin.
Since the muscle contracts slowly and relaxes slowly, a contact
time of 90 seconds and 5 min time cycle are followed to record
DRCs of stock and test solutions of serotonin.
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12.  Select two doses of standard form the DRC of stock solution of
serotonin eliciting sub-maximal responses and bearing a dose
ratio 1:2 preferentially and designate them as n1 and n2 elicits the
responses S1 and S2 respectively .
 Select one dose of test from the DRC of test solution in such a
way that the response due to this dose lies preferentially
between n1 and n2 and designate it as t elicits the response T.
 Record 3 sets of responses due to n1, n2 and t adding them to
organ bath in a randomized fashion as per Latin square design
mentioned below. The Latin square design of addition of doses is
followed to ensure good randomization and to account for the
fluctuating sensitivity of the tissue.
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n1 n2 t
n2 t n1
t n1 n2
Latin Square Design -Three point bioassay

13.  Measure the height of various responses to calculate the mean
height of each response (Mean of S1, S2, and T)
 Calculate the unknown concentration of serotonin (T) using the
following formula and interpret the result.
Where, n1 = Lower standard dose
n2 = Higher standard dose
t = Test dose
S1 = Mean of responses of n1
S2 = Mean of responses of n2
T = Mean of responses of t
Cs= concentration of serotonin stock (10 μg/ml)
13

14. Kymographrecordingof contractions
14
Serotonin
(10μg/ml)
0.1 0.2 0.4 0.8 1.6
SubmaximalDose
Ceiling Dose
SupramaximalDose
Part I- Dose Response Curve (DRC) of Serotonin (10 μg/ml)
Responses
Continued
3.2

15. Kymographrecordingof contractions
15
Serotonin
(T)
0.1 0.2 0.4 0.8 1.6
SubmaximalDose
Ceiling Dose
SupramaximalDose
Part II- Dose Response Curve (DRC) of Serotonin (T)
Responses
Continued
3.2

16. Kymograph recording of contractions- continue
16
Part III- Recording of responses of n1, n2 & t in three point bioassay.
n1- Lower standard dose (0.4ml)
n2- Higherstandard dose (0.8 ml)
t- Test dose (0.6ml)
n1 n2 t
n2 t n1
t n1 n2
Latin Square Design
Three point bioassay

17. Observation table- Example
17
Sr.
No.
Drug
(Conc.)
Dose
(ml)
Dose
(μg)
log
(dose)=
log (dose
in μg )
Response in Conc. of Serotonin in
40 ml organ bath=
Dose (μg)/ Bath
Capacity (ml)
Height
(cm/mm)
%
1
Serotonin
(10 μg/ml )
0.1 1 0 0.3 16.67 0.025
2 0.2 2 0.3010 0.5 27.78 0.05
3 0.4 4 0.6021 1.4 77.78 0.1
4 0.8 8 0.9031 1.6 88.89 0.2
5 1.6 16 1.2041 1.8 100.00 0.4
6 3.2 32 1.5051 1.8 100.00 0.8
Part I- Dose Response Curve (DRC) of Serotonin (10 μg/ml)
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 0.5 1 1.5 2 2.5 3
%
RESPONSE
LOG(DOSE)
Log (DRC)of Serotonin Stock (10 μg/ml)

18. Observation table- Example
18
Sr.
No.
Drug
(Conc.)
Dose
(ml)
Response in
Height
(cm/mm)
1 n1 0.4
2 n2 0.8
3 t 0.6
4 n2 0.8
5 t 0.6
6 n1 0.4
7 t 0.6
8 n1 0.4
9 n2 0.8
Part II- Dose Response Curve (DRC) of Serotonin (T)
(In the way as that of Serotonin stock )
Part III- Recording of responses of n1, n2 & t in three point bioassay.

19. Calculation
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Magnificationvalue (Mf) = d (F-W)/ d (F-T)
Where-
d (F-W) → distance between fulcrumand stylus (writing tip)
d (F-T) → distance between fulcrumand point of attachment of tissue
Unknown concentration of Serotonin T (μg/ml)
Calculate mean of heightsof standard and test responses due to n1, n2 and t
Unknown concentrationof Serotonin T (μg/ml)

20. Result and interpretation
 The concentration response curves (CRCs) or dose response
curves (DRCs) of serotonin stock (10 μg/ml) and test solution
using isolated rat fundus strip preparation was recorded and
interpreted graphically as sigmoid or S-shape curves.
 The unknown concentration of serotonin (T) using isolated
rat fundus strip preparation by three point bioassay was
found to be .........μg/ml.
 The magnification value was found to be-.........
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21. 21
Thank You...