Objective Principle Requirements Experimental specifications (conditions) Preparation of histamine standard solution Preparation of Tyrode solution (PSS) Procedure Kymograph recording of contractions Observation table Calculation Result and interpretation

1. Experiment No. 6
Bioassay of histamine using guinea pig
ileum by matching method
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 histamine standard solution
 Preparation of Tyrode solution (PSS)
 Procedure
 Kymograph recording of contractions
 Observation table
 Calculation
 Result and interpretation
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3. Objective
To determine unknown concentration of histamine using
isolated guinea pig ileum by matching bioassay.
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4. Principle
It is the simplest type of bioassay.
In this type of bioassay, the response of the test substance is
taken first and the observed response is tried to match with
the response that is obtained with the standard drug.
Several responses of the standard drug are recorded till a
close matching response to that of test substance is
observed and the corresponding concentration is thus
calculated.
This assay is employed when the sample size is very small.
Since the assay does not involve the recording of
concentration response curve (CRC), the sensitivity of the
preparation is not taken into consideration. Therefore, the
precision and reliability of such bioassay is not good.
Matching Bioassay
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5. Principle
Histamine is an autocoid having profound physiological
effect in the body. Besides the triple response caused, it
has spasmogenic response on the intestinal smooth
muscle. By acting on H1- histamine receptors, it causes
contraction of intestinal smooth muscle.
Guinea pig is highly sensitive to histamine. The guinea pig
ileum preparation is very commonly used for isolated
tissue experiments. It is sensitive to even ng/ml
concentrations of histamine.
Overnight fasted guinea pigs are used to record better
response of histamine on intestinal smooth muscle.
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6. Requirements
Animal: Guinea pig (400-600 g, overnight fasted)
Physiological solution: Tyrode solution.
Drug- Histamine (Stock solution: 1 mg/ml), Histamine std. (1
or 10 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. Simple straw lever or 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 guinea pig ileum preparation
 Drug- Histamine (Stock solution: 1 mg/ml), Histamine std. (1 or
10 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 histamine standard solution
Stock solution- 1 mg/ml or 1000 μg/ml
 Dissolve 1 mg of histamine in 1 ml of distilled water →
1000 μg/ml
Standard solution
 Dilute 0.1 ml of 1000 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
 Guinea pig 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 the contraction due to histamine test dose (T) and
selected dose of standard (S) (of either 1 or 10 μg/ml) using
frontal writing lever. Contact time of 90 seconds and 5 minutes
of total time cycle may be followed for recording of response of
T and S.
 Observe the doublet of response (T and S) and decide whether
to increase or decrease the dose of S till a close matching
response of S to that of T is obtained. Matching of responses
should be ensure simply by measuring the height of the
responses (cm/ mm).
 Record the doubled dose response of the doses of standard and
test which shows the perfect match.
 Properly label and fix the kymograph recording of responses
with the help of fixing solution.
 Calculate the corresponding concentration of histamine in test
dose (T) is then using the formula-
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12. Kymograph recording of contractions
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T1.0 S0.2 S0.4
Doubled Dose
Response
Perfect Match
Recording of responses of test dose (T) and standard
dose (S) of Histamine in Matching Bioassay
Fig. Bioassay of histamine using guinea pig ileum by matching method
T1.0 T1.0 S0.3 T2.0 S0.6

13. Observation table- Example
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Sr.
No.
Drug
(Conc.)
Dose
(ml)
Response in
Height (cm/mm)
1 T 1.0 2.5
2 S (10 μg/ml) 0.2 1.8
3 T 1.0 2.5
4 S (10 μg/ml) 0.4 3.0
5 T 1.0 2.5
6 S (10 μg/ml) 0.3 2.5
7 T 2.0 3.5
8 S (10 μg/ml) 0.6 3.5

14. 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 Histamine T (μg/ml)
Conc. of Unknown (T) = (0.3/1) X 10 = 3.0 μg/ml
Conc. of Unknown (T) = (0.6/2) X 10 = 3.0 μg/ml
OR

15. Result and interpretation
 The unknown concentration of histamine (T) using isolated
guinea pig ileum by matching bioassay was found to be
.........μg/ml.
 The magnification value was found to be-.........
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Thank You...