This document describes procedures for determining the unknown concentration of acetylcholine (ACh) using isolated tissue preparations from rats, including the rat ascending/descending colon, uterus, anococcygeus muscle, and vas deferens. Each procedure involves sacrificing the rat, isolating the tissue, mounting it in an organ bath with physiological salt solution, applying standard and test doses of ACh to elicit a response, and using an experimental design such as matching or bracketing to calculate the unknown concentration.

1. Acetylcholine bioassay
Dr. S. Parasuraman, M.Pharm., Ph.D.,
Associate Professor & Unit Head,
Unit of Pharmacology,
Faculty of Pharmacy, AIMST University,
Bedong 08100, Malaysia.

2. Acetylcholine bioassay
• Exp 1: Determine unknown concentration of acetylcholine (ACh) using
rat ascending/descending colon
• Exp 2: Determine unknown concentration of acetylcholine (ACh) using
rat uterus
• Exp 3: Determine unknown concentration of acetylcholine (ACh) using
rat anococcygeus muscle preparation
• Exp 4: Determine unknown concentration of acetylcholine (ACh) using
rat vas deferens

3. Determine unknown concentration of acetylcholine
(ACh) using rat ascending/descending colon
•Materials
• Animal/tissue : Rat/Ascending (AC) or descending colon (DC)
• PSS : Krebs (AC) /deJalon (DC)
• Lever : Frontal writing
• Magnification : 4-7x
• Tension/load : 2g
• Air : O2 or Carbogen
• Temperature : 25°C (AC)/37°C (DC)
• Drug : Acetylcholine chloride (ACh)

4. Determine unknown concentration of acetylcholine
(ACh) using rat ascending/descending colon
• Method
• Step I: Keep the animal (rat) fasting for at
least 24 hours
• Step II: Sacrifice the rat by stunning,
dissect the animals and identify the
colon (ascending/descending)
• Step III: Cut small piece (1.5-3 cm) as per
the inner organ bath volume and clean it
with a lukewarm water or PSS with the
help of a syringe.

5. Determine unknown concentration of acetylcholine
(ACh) using rat ascending/descending colon
• Method
• Step IV: Then tie both the ends with the help of nylon or cotton thread and tie it
to the balanced horizontal lever.
• Step V: Leave the tissue at least for 30 min for relaxation.
• Step VI: Then, take the response with the standard and test drug (prepared by
serial dilution).
• Step VII: Select the experimental design and take the responses of the drug.
• Step VIII: Calculate recorded responses, according to the selected experimental
design.
• Note: The ascending/descending colon is not the suitable tissue for the assay
of ACh, whereas it may give erratic responses. Intestine preparations are very
commonly used in the isolated tissue experiment due to the ease of isolation;
more handling resistant than other tissues which have variable spontaneous
responses.

6. Determine unknown concentration of acetylcholine
(ACh) using rat uterus
•Materials
• Animal/tissue : Female rat/uterus
• PSS : Krebs/deJalon/McEwen
• Lever : Frontal writing
• Magnification : 4-7x
• Tension : 1-4g
• Air : O2 or Carbogen
• Temperature : 32-37°C (at 32°C spontaneous
responses are low)
• Drug : Acetylcholine chloride (ACh)

7. Determine unknown concentration of acetylcholine
(ACh) using rat uterus
• Method
• Step I: Female rat (FR) weighing
around 150-300 g is taken, and 24
hr prior to the experiment, the rat is
primed with 0.1 mg/kg stilbestrol,
IM.
• Step II: Sacrifice the rat by stunning,
Identify the vaginal orifice. dissect
the animals to identify two horns of
the uterus. Cut the ONE horns (1.5-
3 cm) as per the inner organ bath
volume.

8. Determine unknown concentration of acetylcholine
(ACh) using rat uterus
• Method
• Step III: Select the experimental design (matching, bracketing,
3-point, or 4point assay).
• Step IV: Then, take the response with the standard and test
drug (prepared by serial dilution).
• Step V: Select the experimental design and take the responses
of the drug.
• Step VI: Calculate recorded responses, according to the
selected experimental design.
• Note: The response of uterus preparation mainly depends on the
animal age due to variation in the estrus cycle.

9. Determine unknown concentration of acetylcholine
(ACh) using rat anococcygeus muscle preparation
• Materials
• Animal/tissue : Rat/anococcygeus muscle
• PSS : Krebs
• Lever : Frontal writing
• Magnification : 7-10x
• Tension : up to 1 gm
• Air : O2 or Carbogen
• Temperature : 35-37°C
• Drug : Acetylcholine chloride

10. Determine unknown concentration of acetylcholine
(ACh) using rat anococcygeus muscle preparation
• Method
• Step I: Sacrifice the rat by stunning, cut open the abdomen of rat by a vertical
midline incision, and then exposed the abdominal viscera.
• Step II: Identify the root of rectum interiorly and then identify the 2 thin
strips of anococcygeus muscle which arise from the vertebrae and meet at
the terminal part of colon (anus).
• Step III: Cut the rectal colon 2-3 cm from the anus and clear from the
connective tissues and isolate the 2 strips of anococcygeus muscle.
• Step IV: Keep in PSS and supply continuous constant air (O2 or carbogen).
• Step V: Select the experimental design (matching, bracketing, 3-point, or
4point assay). Prepare the standard and test drugs and select the
experimental design. Calculate recorded responses, according to the selected
experimental design.

11. Determine unknown concentration of acetylcholine
(ACh) using rat vas deferens
• Materials
• Animal/tissue : Rat/vas deferens
• PSS : Krebs/Ringer/Tyrode/ McEwen
• Lever : Frontal writing
• Magnification : 6-8x
• Tension : 0.3-1g
• Air : O2 or Carbogen
• Temperature : 32-37°C
• Drug : Acetylcholine chloride

12. Determine unknown concentration of acetylcholine
(ACh) using rat vas deferens
• Method
• Step I: Sacrifice the rat by stunning, Cut open the abdomen of rat by a
vertical midline incision, and then expose the abdominal viscera.
Identify the testes then pull it to one side and vas deferens, freed out
from the epididymis. Cut into desired length of tissue.

13. Determine unknown concentration of acetylcholine
(ACh) using rat vas deferens
• Method
• Step II: Put into the Petri dish filled with PSS with proper continuous
distinguishable aeration.
• Step III: Put the tissue into the organ bath and perform the
experiment as per selected experimental design (matching, bracketing,
3-point, or 4point assay).
• Step IV: Prepare the standard and test drugs and select the experimental
design.
• Step V: Calculate recorded responses, according to the selected
experimental design.