Objective Principle Requirements Experimental specifications (conditions) Preparation of ACh stock and standard solutions Preparation of frog ringer solution (PSS) Procedure Kymograph recording of contractions Observation table Calculation Result and interpretation

1. Experiment No. 7
Bioassay of acetylcholine using rat ileum
by four 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 ACh stock and standard solutions
• Preparation of frog ringer solution (PSS)
• Procedure
• Kymograph recording of contractions
• Observation table
• Calculation
• Result and interpretation
2

3. Objective
To determine unknown concentration of acetylcholine
(ACh) using isolated rat ileum preparation by four
point bioassay.
3

4. Four point bioassay
4
In such a type of multiple point bioassay, two responses of
standard drug and two responses of test drug are recorded.
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 selection of test response is determined
by hit and trial method so that the responses fall on the
linear part of the curve.
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 ileum or colon are the smooth muscles which are sensitive
to ACh and shows contraction.
Mechanism of smooth muscle contraction
ACh acts on muscarinic M3- subtype of 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 smooth muscle contraction.
Overnight fasted rats are used to record better response of
ACh on intestinal smooth muscles such as ileum and colon.

6. Requirements
Animal: Albino rats (150-200 g, overnight fasted)
Physiological solution: Tyrode solution.
Drug- ACh (Stock solution: 1 mg/ml), ACh std. (1 or 10 or 100
μg/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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7. Experimental specifications (conditions)
 Isolated tissue- Isolated rat ileum preparation
 Drug- ACh (Stock solution: 1 mg/ml), ACh std. (1/ 10/ 100
μg/ml)
 Physiological salt solution (PSS)- Tyrode solution.
 Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact
time- 90 seconds, Washing period- 3 minutes
 Applied load/ tension- 0.5 g
 Bath capacity- 40 ml
 Bath temperature- 32- 35°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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8. Preparation of ACh stock and standard solutions
Stock solution- 1 mg/ml or 1000 μg/ml
 Dissolve 1 mg of ACh in 1 ml of distilled water → 1000 μg/ml
Standard solutions
 Dilute 1 ml of stock solution up to 10 ml with distilled water
→ 100 μg/ml
 Dilute 1 ml of 100 μg/ml solution up to 10 ml with distilled water
→ 10 μg/ml
 Dilute 1 ml of 10 μg/ml solution up to 10 ml with distilled water
→ 1 μg/ml
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9. Preparation of Tyrode solution (PSS)
 Prepare 1 litre of Tyrode solution by dissolving NaCl (8.0
g), KCl (0.2 g), MgCl2 (0.1 g), NaHCO3 (1.0 g), NaH2PO4
(0.05 g) and glucose (2.0 g) in distilled water.
 MgCl2 should be added at last.
 CaCl2 (0.2 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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10. Procedure
 Albino rat is sacrificed by a blow on the head and carotid
bleeding.
 Cut open the abdomen and lift the caecum to trace the ileocecal
junction. Cut and remove a few centimeter long of the ileal
portion and immediately place it in the watch glass containing
Tyrode solution. Trim the mesentery and with gentle care clean
the contents of the ileum by pushing the Tyrode solution into
the lumen of the ileum. Utmost care should be taken to avoid
any damage to the gut muscle. Cut the ileum into small
segments of 2-3 cm long.
 Take a piece of ileum of 2-3 cm long and tie the thread to the top
and the bottom ends without closing the lumen, and mount the
tissue in the organ bath containing Tyrode solution maintained
at 32- 35°C and bubbled with O2 or air. A tension of 0.5 g is
applied and the tissue is allowed to equilibrate for 30 minutes
before adding the drugs to the organ bath.
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11.  Record graded responses (CRC/ DRC) with standard solution of
ACh until the peak effect is obtained.
 Select two concentrations (A, B) of the standard ACh eliciting
submaximal responses (S1, S2) and bearing a dose ratio 1:2
preferentially.
 Select two suitable volumes of test solution by trial and error
method in such a way that the response T1 due to lower dose of
test (C) lies preferentially between S1 and S2. The higher volume
of test solution selected would be D such that the dose ratio
B/A= D/C and the response of D is T2. All the four responses (S1,
S2, T1 , T2) due to the doses thus selected (A, B, C, D) must lie on
the linear part of the standard (sigmoid) curve.
 Record four sets of responses due to A, B, C and D adding them
to the organ bath in a random fashion (Latin square design) as
shown.
 Properly label and fix the kymograph recording of responses
with the help of fixing solution.
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12.  Measure various responses to calculate mean of each response
(Mean of S1, S2, T1 and T2).
 Calculate the potency ratio (M) using the following formula-
Where, A= Lower volume of the standard drug,
B= Higher volume of the standard drug,
C= Lower volume of the test drug, and
= Mean standard and test responses.
 Determine the strength of unknown solution of ACh using the
concentration of the standard ACh solution and potency ratio
(M). (Simply multiply potency ratio, M with standard
concentration of ACh, Cs).
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13. Kymograph recording of contractions
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ACh Std.
(100 μg/ml)
0.1 0.2 0.4 0.8 1.6
Submaximal Dose
Ceiling Dose
Supramaximal Dose
Part I- Dose Response Curve (DRC) of ACh Std.
Responses
Continued
3.2

14. Kymograph recording of contractions- continue
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Part II- Recording of responses of A, B, C & D in four point bioassay.
Lower volume of the standard drug (0.4ml)
Higher volume of the standard drug (0.8 ml)
Lower volume of the test drug (0.6ml)
Higher volume of the test drug (1.2 ml)
A B C D
B C D A
C D A B
D A B C
Latin Square Design
Four point bioassay

15. Observation table- Example
15
Sr.
No.
Drug
(Conc.)
Dose
(ml)
Dose
(μg)
log
(dose)=
log (dose
in μg )
Response in Conc. of ACh in 40 ml
organ bath=
Dose (μg)/ Bath
Capacity (ml)
Height
(cm/mm)
%
1
ACh
(100 μg/ml )
0.1 10 1.0 0.3 16.67 0.25
2 0.2 20 1.3010 0.5 27.78 0.5
3 0.4 40 1.6021 1.4 77.78 1
4 0.8 80 1.9031 1.8 100.00 2
5 1.6 160 2.2041 1.8 100.00 4
6 3.2 320 2.5051 1.8 100.00 8
Part I- Dose Response Curve (DRC) of ACh Std.
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 ACh Standard (100μg/ml)

16. Observation table- Example
16
Sr.
No.
Drug
(Conc.)
Dose
(ml)
Response in
Height
(cm/mm)
1 A 0.4
2 B 0.8
3 C 0.6
4 D 1.2
5 B 0.8
6 C 0.6
7 D 1.2
8 A 0.4
Part II- Recording of responses of A, B, C & D in four point bioassay.
Sr.
No.
Drug
(Conc.)
Dose
(ml)
Response in
Height
(cm/mm)
9 C 0.6
10 D 1.2
11 A 0.4
12 B 0.8
13 D 1.2
14 A 0.4
15 B 0.8
16 C 0.6

17. Calculation
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Magnification value (Mf) = d (F-W)/ d (F-T)
Where-
d (F-W) → distance between fulcrum and stylus (writing tip)
d (F-T) → distance between fulcrum and point of attachment of tissue
Unknown concentration of ACh T (μg/ml)
Calculate mean of heights of standard and test responses due to A, B, C and D
Unknown concentration of ACh T (μg/ml)= Potency Ratio (M) X Cs

18. Result and interpretation
 The concentration response curve (CRC) or dose response
curve (DRC) of standard concentration of ACh (100 μg/ml)
by using isolated rat ileum preparation was recorded and
interpreted graphically as sigmoid or S-shape curve.
 The unknown concentration of ACh (T) using isolated rat
ileum preparation by four point bioassay was found to be
.........μg/ml.
 The magnification value was found to be-.........
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19. 19
Thank You...