ALTERNATIVES TO ANIMAL EXPERIMENTATION MANUAL

B. Pharm. Part-III, Sem.-VI

(Pharmacology-III, PH-3403)

Name: Roll No.:

ALTERNATIVES TO ANIMAL EXPERIMENT LABORATORY DEPARTMENT OF PHARMACEUTICS INDIAN INSTITUTE OF TECHNOLOGY

(BANARAS HINDU UNIVERSITY) VARANASI-221 005

2014

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Laboratory Directions and Instructions to the Students

1. Purpose of Laboratory Meetings

: Laboratory exercises, experiments and demonstrations provide opportunities to observe what you heard about in lectures and read about in texts. A sincere interest in laboratory procedures will often be the key to understanding some problem. Take notes on the instructor’s preliminary instructions and discussions.

2. Completion of Laboratory Exercises

: Exercises are to be completed during the laboratory period assigned to them, unless and otherwise designated by the teacher. All work should be kept in your manual and used as study material for examination, and for future reference.

3. Attendance

: You are responsible for work missed, so arrange with your teacher to make it up as soon as possible.

4. Laboratory Etiquette and Procedures

: Be in your place and ready for class at the beginning of the period.

5. Do not disturb equipment and demonstrations that have been set up, until you have been told how to proceed with the experiment.

6. Pay careful attention to directions given by the laboratory instructor so that you will

know how to make the best use of your time and materials.

7. Be considerate of your laboratory associates. Your activities may disturb others so much that they will get very little out of their laboratory works.

8. As you are going to work in a batch, do your full share.

9. At the end of the period leave your place in good working order.

10. Bring all necessary items :

(a) Foot ruler or centimeter scale. (b) Eraser. (c) Soft and hard pencils.

11. Read over the laboratory material for the text laboratory meeting each time before you

come to class.

12. Think through your results carefully, so that any conclusions which you may draw will be based on sound, logical reasoning and not on hasty, ill–formed ideas.

13. Submit this manual after completion of the ‘practical scheme’ for overall evaluation.

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INDEX

S. No.

Experiment

Page No.

Date

Teacher’s Signature

1. To study anatomy and physiology of human digestive system.

3 - 7

2. To study the effect of Physostigmine on the Dose Response Curve (DRC) of Acetylcholine using isolated frog rectus muscle through X-COLOGY simulated software.

8 – 11

3. To study the effect of d-Tubocurarine on the Dose Response Curve (DRC) of Acetylcholine using isolated frog rectus muscle through X-COLOGY simulated software.

12 - 13

4. To study the effect of Atropine on the Dose Response Curve of Acetylcholine using isolated rat ileum through X-COLOGY simulated software.

14 – 16

5. To perform bioassay of Histamine by three point assay method on isolated guinea pig ileum through X-COLOGY simulated software.

17 - 19

6. To perform bioassay of Histamine by matching assay method on isolated guinea pig ileum through ExPharm simulated software.

21 - 23

7. To perform bioassay of Oxytocin by interpolation method on isolated rat uterus through X-COLOGY simulated software.

24 - 25

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EXPERIMENT NO. 01

OBJECTIVE: To study anatomy and physiology of human digestive system.

INTRODUCTION:

The digestive system is the system by which ingested food is acted upon by physical and

chemical means to provide the body with nutrients it can absorb and to excrete waste products. In

mammals the system includes the alimentary canal extending from the mouth to the anus, and the

hormones and enzymes assisting in digestion.

In an adult male human, the GI tract is approximately 5 meters long and consists of the upper and

lower GI tracts. The tract may also be divided into foregut, midgut, and hindgut, reflecting the

embryological origin of each segment of the tract.

The upper gastrointestinal tract consists of the mouth cavity, salivary glands, pharynx, esophagus, stomach and duodenum.

Upper gastrointestinal tract

The lower gastrointestinal tract comprises the most of the intestines and the anus.

Lower gastrointestinal tract

Bowel or intestine

1. Small intestine, which has three parts:

Duodenum - Here the digestive juices from pancreas and liver mix together Jejunum - It is the midsection of the intestine, connecting duodenum to

ileum. Ileum - It has villi in where all soluble molecules are absorbed into the blood.

2. Large intestine, which has three parts:

Cecum (the vermiform appendix is attached to the cecum). Colon (ascending colon, transverse colon, descending colon and sigmoid

flexure) Rectum

3. Anus

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Accessory organs to the alimentary canal include the liver, gallbladder, and pancreas. The liver

secretes bile into the small intestine via the bile duct, employing the gallbladder as a reservoir.

Apart from storing and concentrating bile, the gallbladder has no other specific function. The

Accessory organs

pancreas secretes an isosmotic fluid containing bicarbonate, which helps neutralize the acidic

chyme, and several enzymes, including trypsin, chymotrypsin, lipase, and pancreatic amylase, as

well as nucleolytic enzymes (deoxyribonuclease and ribonuclease), into the small intestine. Both

of these secretory organs aid in digestion.

The gastrointestinal tract has a uniform general histology with some differences that reflect the

specialization in functional anatomy. The GI tract can be divided into four concentric layers:

Histology

• Mucosa • Submucosa • Muscularis externa (the external muscle layer) • Adventitia or serosa

The mucosa is the innermost layer of the gastrointestinal tract that is surrounding the

Mucosa

lumen, or

space within the tube. This layer comes in direct contact with food (or bolus), and is responsible

for absorption and secretion, important processes in digestion.

The mucosa can be divided into:

• Epithelium • Lamina propria • Muscularis mucosae

The submucosa consists of a dense irregular layer of connective tissue with large blood vessels,

lymphatics, and nerves branching into the mucosa and muscularis externa.

Submucosa

The muscularis externa consists of an inner circular layer and an outer longitudinal muscular

layer. The circular muscle layer prevents food from traveling backward and the longitudinal layer

Muscularis externa

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shortens the tract. The coordinated contraction of these layers is called peristalsis and propels the

bolus, or balled-up food, through the GI tract.

The

Adventitia

adventitia consists of several layers of epithelia.

Human digestion process

In humans, digestion begins in the

Oral cavity

oral cavity where food is chewed. Saliva is secreted in large

amounts (1-1.5 liters/day) by three pairs of exocrine salivary glands (parotid, submandibular, and

sublingual) in the oral cavity, and is mixed with the chewed food by the tongue. The saliva serves

to clean the oral cavity and moisten the food, and contains digestive enzymes such as salivary

amylase, which aids in the chemical breakdown of polysaccharides such as starch into

disaccharides such as maltose. It also contains mucin, a glycoprotein which helps soften the food

into a bolus.

Food enters the stomach through the cardiac orifice where it is further broken apart and

thoroughly mixed with gastric acid and pepsin to break down proteins. The acid itself does not

break down food molecules, rather it provides an optimum pH (3.5) for the reaction of the

enzyme

Stomach

pepsin and kills many microorganisms that are ingested with the food. It can also

denature proteins. This is the process of reducing polypeptide bonds and disrupting salt bridges

which in turn causes a loss of secondary, tertiary or quaternary protein structure. The parietal cells

of the stomach also secrete a glycoprotein called intrinsic factor which enables the absorption of

vitamin B-12. Other small molecules such as alcohol are absorbed in the stomach, passing

through the membrane of the stomach and entering the circulatory system directly. Food in the

stomach is in semi-liquid form, which upon completion is known as chyme.

After being processed in the stomach, food is passed to the

Small intestine

small intestine via the pyloric

sphincter. The majority of digestion and absorption occurs here after the milky chyme enters the

duodenum. Here it is further mixed with three different liquids:

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Bile, which emulsifies fats to allow absorption, neutralizes the chyme and is used to

excrete waste products such as bilin and bile acids.

Pancreatic juice made by the pancreas.

Intestinal enzymes of the alkaline mucosal membranes. The enzymes include maltase,

lactase and sucrase (all three of which process only sugars), trypsin and chymotrypsin.

As the pH level changes in the small intestines and gradually becomes basic, more enzymes are

activated further that chemically break down various nutrients into smaller molecules to allow

absorption into the circulatory or lymphatic systems. Small, finger-like structures called villi,

each of which is covered with even smaller hair-like structures called microvilli improve the

absorption of nutrients by increasing the surface area of the intestine and enhancing speed at

which nutrients are absorbed. Blood containing the absorbed nutrients is carried away from the

small intestine via the hepatic portal vein and goes to the liver for filtering, removal of toxins, and

nutrient processing.

After the food has been passed through the small intestine, the food enters the

Large intestine

large intestine.

Within it, digest is retained long enough to allow fermentation due to the action of gut bacteria,

which breaks down some of the substances which remain after processing in the small intestine;

some of the breakdown products are absorbed. In humans, these include most complex

saccharides (at most three disaccharides are digestible in humans). In addition, in many

vertebrates, the large intestine resorbs fluid; in a few, with desert lifestyles, this resorption makes

continued existence possible.

In humans, the large intestine is roughly 1.5 meters long, with three parts: the cecum at the

junction with the small intestine, the colon, and the rectum. The colon it self has four parts: the

ascending colon, the transverse colon, the descending colon, and the sigmoid colon. The large

intestine absorbs water from the bolus and stores feces until it can be egested. Food products that

cannot go through the villi, such as cellulose (dietary fiber), are mixed with other waste products

from the body and become hard and concentrated feces. The feces is stored in the rectum for a

certain period and then the stored feces is eliminated from the body due to the contraction and

relaxation through the anus. The exit of this waste material is regulated by the anal sphincter.

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EXPERIMENT NO. 02

OBJECTIVE: To study the effect of Physostigmine on the Dose Response Curve (DRC) of

Acetylcholine using isolated frog rectus muscle through X-COLOGY simulated software.

A. SETUP FOR ISOLATED TISSUE PREPARATIONS:

1. Adjust the speed of Sherrington’s Revolving Drum Machine to 0.12 mm/sec. Level the

machine horizontally with the help of basal screws.

2. Adjust the kymograph pasted drum in such a way that the complete deflection of the lever

is recorded on the kymograph.

3. For isolated tissues of warm blooded animals, the outer jacket of Student’s Organ bath is

filled with tap water and thermostat is adjusted to 37°C and the heater is put on (This

setting is not required for the frog related experiments).

4. Adjust the level of Physiological salt solution in the organ tube to 20 ml and mark this level

(This level is kept constant throughout the experiment. This is required to monitor the

concentration of drug being added to the organ tube. Also exact concentration of drug

coming in contact with the tissue can be determined if this level is fixed at the time of

adding the drug).

5. Thread the suturing needle.

6. Balance the lever and adjust the weight on the tissue. For skeletal muscle like frog rectus

abdominis put 1 g load on the lever. For smooth muscle preparations like rat ileum adjust

500 mg load on the lever.

7. Keep aeration rate at one bubble per second.

8. Other instruments required:

Dissection box

Thread

1 ml Tuberculin syringe

Suturing needles

Infusion set

B. DISSECTION:

Pithing of frog: Insert a sharp needle in the foramen magnum towards the brain and destroy a

part of it. Then remove and reinsert the needle in the open spinal canal and destroy part of spinal

cord by inserting the needle backwards. This may cause the frog to urinate and throw its hind legs

in convulsion.

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Checking the reflex: To see whether the frog has been properly pithed, touch the cornea of eye

with the needle and see whether corneal responses have completely subsided. Also, “touch and

pain” reflex can be checked by superficially pricking the hind legs of the frog to see whether

jerking movement occurs. A properly pithed frog shows neither corneal nor pain reflex.

Lay the pithed frog on its back. With a fine scissor, take a small ‘V’ shaped cut in the

abdominal skin at the pelvic girdle. Insert a curved scissor, in the ‘V’ shaped incision and

cut the abdominal skin up to pectoral girdle.

The underlying muscular part show rectus abdominis muscle. Take a bold cut on one side

of the central vein near pelvic girdle. Through this cut, insert the blunt side of the scissor

and take a cut up to pectoral girdle without injuring the visceral organs.

Isolate a piece of about 0.5 cm width on one side of central vein.

Take the isolated muscle in a petri-plate containing aerated Ringer solution. Pass a piece of

thread through one end of the muscle and put a knot. Tie the end having shorter thread to

the aeration tube. Hold the longer thread along with aeration tube and transfer into the

organ tube. Take care to avoid any accidental stretch on the muscle.

Tie the longer thread to the lever. Maintain the tissue as such for at least 45 minutes to get

stabilized in the in-vitro atmosphere. During this time give at least three washings to the

tissue (the tissue is washer either by draining out the physiological salt solution and

refilling the organ tube or it can be washed by overflowing the physiological salt solution

through the organ tube).

Recording of DRC of Acetylcholine:

Take the dose-response relationship by administering varying doses of acetylcholine. For

the convenience of plotting the dose-response curve, increase the doses either in the

geometric progression (2, 4, 8, 16 etc.) or by logarithmic intervals (1, 3, 10, 30, 100 etc.).

Continue taking response until maximum response is achieved i.e. two consecutive doses

give equal response. Take care to wash the tissue after each dose.

Perfuse the tissue with physiological salt solution containing Physostigmine (2 µg/ml) and

again take the dose-response relationship of Acetylcholine as earlier. Tabulate the

observations in the following table.

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Effect of Physostigmine on the DRC of Acetylcholine using frog rectus muscle

Dose of Acetylcholine (ml)

0.02 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4

Quantity of drug added (µg)

0.2 0.5 1 2 4 8 16 32 64

Log Concentration -0.7 -0.3 0 0.3 0.6 0.9 1.2 1.5 1.8

Response in absence of Physotigmine (mm)

% Response (% of maximum response)

Response in presence of Physotigmine (mm)

% Response (% of maximum response)

INFERENCE:

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EXPERIMENT NO. 03

OBJECTIVE: To study the effect of d-Tubocurarine on the Dose Response Curve (DRC) of

Acetylcholine using isolated frog rectus muscle through X-COLOGY simulated software.

A. SETUP FOR ISOLATED TISSUE PREPARATIONS: As in Experiment number 02.

B. DISSECTION: As in Experiment number 02.

Recording of DRC of Acetylcholine:

Take the dose-response relationship by administering varying doses of acetylcholine. For

the convenience of plotting the dose-response curve, increase the doses either in the

geometric progression (2, 4, 8, 16 etc.) or by logarithmic intervals (1, 3, 10, 30, 100 etc.).

Continue taking response until maximum response is achieved i.e. two consecutive doses

give equal response. Take care to wash the tissue after each dose.

Perfuse the tissue with physiological salt solution containing d-Tubocurarine (2 µg/ml) and

repeat the entire procedure as earlier. Tabulate the observations as follows:

Effect of d-Tubocurarine on the DRC of Acetylcholine using frog rectus muscle

Dose of Acetylcholine (ml)

0.02 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4

Quantity of drug added (µg)

0.2 0.5 1 2 4 8 16 32 64

Log Concentration -0.7 -0.3 0 0.3 0.6 0.9 1.2 1.5 1.8

Response in absence of d-Tubocurarine (mm)

% Response (% of maximum response)

Response in presence of d-Tubocurarine (mm)

% Response (% of maximum response)

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INFERENCE:

14

EXPERIMENT NO. 04

OBJECTIVE: To study the effect of Atropine on the Dose Response Curve of Acetylcholine

using isolated rat ileum through X-COLOGY simulated software.

A. SETUP FOR ISOLATED TISSUE PREPARATIONS: As in Experiment number 02.

Kymograph speed: 0.25 mm/sec

B. ISOLATION AND MOUNTING OF ILEUM:

Give a midline incision on the abdomen. Expose the ileocecal junction. Isolate the ileum

excluding terminal 5 cm. Cut the ileum into pieces of 2-4 cm length and maintain in

warm Tyrode solution (37°C) with aeration.

Take a piece of ileum in a petri-plate containing aerated Tyrode solution. Remove the

mesenteric attachment. Clean the lumen of ileum by passing warm tyrode solution

through the lumen with the help of a pipette (care must be taken not to expose the tissue

to very high distortion which can affect the sensitivity of the tissue to drug substances).

Pass the threaded suturing needle through the wall of the ileum and put a knot. Similarly

pass a thread through another end of the tissue and put a knot.

Hold the thread of the free end along with the aeration tube and carefully transfer the

aeration tube along with the tissue to the organ tube containing warm tyrode solution.

Tie the free end of the tissue to the lever. Allow the tissue to acclimatize for about 30

minutes. During this period give at least 2-3 washes to the tissue.

Recording of DRC of Acetylcholine:

Take the dose-response relationship by administering varying doses of acetylcholine. For

the convenience of plotting the dose-response curve, increase the doses either in the

geometric progression (2, 4, 8, 16 etc.) or by logarithmic intervals (1, 3, 10, 30, 100 etc.).

Continue taking response until maximum response is achieved i.e. two consecutive doses

give equal response. Take care to wash the tissue after each dose.

Perfuse the tissue with Tyrode solution containing Atropine (2 µg/ml) and repeat the

entire procedure as earlier. Tabulate the observations as follows:

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Effect of Atropine on the DRC of Acetylcholine using rat ileum muscle

Dose of Acetylcholine (ml)

0.02 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4

Quantity of drug added (µg)

0.2 0.5 1 2 4 8 16 32 64

Log Concentration -0.7 -0.3 0 0.3 0.6 0.9 1.2 1.5 1.8

Response in absence of Atropine (mm)

% Response (% of maximum response)

Response in presence of Atropine (mm)

% Response (% of maximum response)

Plot a DRC of Acetylcholine in absence and presence of Atropine.

INFERENCE:

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EXPERIMENT NO. 05

OBJECTIVE: To perform bioassay of Histamine by three point assay method on isolated guinea

pig ileum through X-COLOGY simulated software.

A. SETUP FOR ISOLATED TISSUE PREPARATIONS: As in Experiment number 02.

Kymograph speed: 0.25 mm/sec

B. ISOLATION AND MOUNTING OF ILEUM:

Give a midline incision on the abdomen. Expose the ileocecal junction. Isolate the ileum

excluding terminal 5 cm. Cut the ileum into pieces of 2-4 cm length and maintain in

warm Tyrode solution (37°C) with aeration.

Take a piece of ileum in a petri-plate containing aerated Tyrode solution. Remove the

mesenteric attachment. Clean the lumen of ileum by passing warm tyrode solution

through the lumen with the help of a pipette (care must be taken not to expose the tissue

to very high distortion which can affect the sensitivity of the tissue to drug substances).

Pass the threaded suturing needle through the wall of the ileum and put a knot. Similarly

pass a thread through another end of the tissue and put a knot.

Hold the thread of the free end along with the aeration tube and carefully transfer the

aeration tube along with the tissue to the organ tube containing warm tyrode solution.

Tie the free end of the tissue to the lever. Allow the tissue to acclimatize for about 30

minutes. During this period give at least 2-3 washes to the tissue.

THREE POINT ASSAY:

Take graded response of standard drug solution

Select two doses of standard solution of which the response (S2) due to higher dose (n2)

is about double the response (S1) due to lower dose (n1) and there is preferably 2:1

dosage ratio

From the graded response of test drug solution select the dose (t) that give response

between (S1) and (S2)

Repeat the doses and take responses as per Latin Square Randomized schedule for dosing

n1, n2, t, n2, t, n1, t, n1, n2

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Table:

Dose of standard (ml)

0.02 0.05 0.1 0.2 0.4 n1 0.8 n2 1.6 3.2

Response (mm)

9 16 22 34 42 (S1) 52 (S2) 60 65

Dose of Test (ml)

0.02 0.05 0.1 0.2 (t)

Response (mm)

14 25 37 48 (T)

Readings as Latin Square Randomization:

Doses n1 (0.4ml) n2 (0.8 ml) t (0.2 ml)

Responses (mm)

42 52 48

41 51 47

43 53 49

Average (mm) S1 = 42 S2 = 52 T = 48

The concentration of the test sample can be found out using following formula

Ct = (n1/t) * Antilog {[(T - S1)/(S2-S1)] * log (n2/n1)}*Cs

Where: n1 & n2 are doses of standard. t is dose of Test S1 and S2 are responses for n1 & n2 respectively T is response for t. Cs & Ct are concentrations of Standard and Test respectively. (Cs = 20 µg/ml, Ct = Concentration of Unknown)

RESULT:

The concentration of histamine in the given sample was found to be ...................... µg/ml.

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EXPERIMENT NO. 06

OBJECTIVE: To perform bioassay of Histamine by matching assay method on isolated guinea

pig ileum through ExPharm simulated software.

Drug & Solution: 1. Histamine 2. Tyrode's solution

Set up: A guinea-pig is sacrificed, abdomen is opened and the ileum is isolated. Mesenteric

attachment is released and the ileum is cut across and placed in Petri dish containing Tyrode's

solution. A small segment of ileum (about 5 cm) is cut and cleaned and one end is fixed to a

tissue holder with the help of a thread. The other end is tied to a thread and the tissue holder along

with the tissue is placed inside an organ bath. Then the thread is attached to a force transducer

which is connected to a physiograph.

The water bath is filled with water and warmed by a heater cum thermostat to maintain a

temperature of 37°C. The organ bath filled with Tyrode's solution is connected to a reservoir. The

organ bath can be emptied by opening the outlet and refilled with Tyrode's solution from

reservoir.

Ileum and tissue holder are placed in the organ bath. The upper end of the ileum is attached to a

force transducer by a long thread. An aerator is connected to the tissue holder. The tissue holder

fixed in position tightly with clamps and attached to an aerator through a rubber tube. The air

bubbles out at the other end inside the Tyrode's solution. Full view of the set up with reservoir

containing Tyrode's solution

Procedure:

1. Choose a dose of histamine and inject. Obtain a dose response curve by increasing the dose in

geometric progression. The starting dose can be 0.1 mcg. If the tissue is very sensitive start

from 0.01 mcg. Drug being injected into the organ bath.

2. Once the maximum response is reached, start the matching assay.

3. When you start the matching assay you will be given a solution of unknown concentration of

histamine. You will have to choose a standard curve (STD) from your dose response curve

and enter the amount of histamine (in mcg) which produced the standard curve. The standard

curve is chosen on the basis that the response produced by the curve is about 50% of maximal

response (maximal response is one which does NOT increase further with an increase in the

dose).

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4. Then inject the std and the unknown alternatively. The unknown is given in ml. The starting

dose can be 0.1 ml. Adjust the dose of unknown until it matches with the std. Matching is

achieved by trial and error.

5. Matching can be confirmed by giving 2s or s/2. (Double or half the amount of std) and half or

double the dose of unknown that matches the std. After matching is confirmed, calculate the

concentration of histamine in the unknown solution.

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EXPERIMENT NO. 07

OBJECTIVE: To perform bioassay of Oxytocin by interpolation method on isolated rat uterus

through X-COLOGY simulated software.

A. SETUP FOR ISOLATED TISSUE PREPARATIONS: As in Experiment number 02.

Kymograph speed: 0.25 mm/sec

B. ISOLATION AND MOUNTING OF RAT UTERUS:

Dissect and open the abdomen. Pull aside the intestine to expose the uterus. Isolate the uterus and

keep it in a Petri dish containing warm De-Jalon’s solution (aerated). Separte on horn of the

uterus and remove the attached fatty tissues.

Pass a threaded suturing needle through one end of the tissue and put a knot. Similarly pass the

thread through another end put a knot. Tie this tissue to aeration tube.

Mount the tissue in to the organ bath and tie the end of longer thread to the lever. Put 500 mg

tension on the tissue. Maintain the bath temperature at 30-32°C and allow the tissue to

acclimatize to in-vitro condition for 20-30 minutes. Give 2-3 washes to tissue during the period.

C. PROCEDURE:

1. Record the DRC of Oxytocin (Standard).

2. Take the response of test solution and calculate the concentration of test solution by

interpolating with standard DRC.

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