Experiment No: 1

Aim: To study in-vitro pharmacology and physiological salt solutions.

Equipment and apparatus required: NA

Learning Objective: To learn the general introduction of in-vitro

pharmacology
 Theory:
➢ Pharmacology is the science which deals with the study of drugs.
➢ The word pharmacology is derived from the Greek words pharmakon means a
“drug or a poison” and logos means “discourse”.
➢ It broadly covers information about the history, source, physicochemical
properties, and mechanism of action, absorption, distribution, metabolism,
excretion and therapeutic uses of drugs.
➢ Drugs are the chemical substances used for the purpose of diagnosis, prevention,
mitigation and cure of the disease in man and or animals.
❑ Main aim of experimental pharmacology:
• Find out the therapeutic agent suitable for human use.
• Study the toxicity of drug
• Study the mechanism and site of action of drug.
✓ In vitro Pharmacology studies the biological effect of a drug in an isolated
environment/tissue, such as cell line or tissue.
✓ In vivo pharmacology is the study of the biological effect of a drug in a
complex living organism.
 ❑ Basic equipment used
Most widely used Instrument in experimental pharmacology is student organ bath assembly in which animal
tissue is placed in order to check the effect of the drug.
This was first designed by Rodolph Magnus in 1904.
The organ bath essentially consists of:
• An outer jacket (water bath) made up of steel, glass or Perspex
• The inner organ or tissue bath made up of glass with a capacity varying from 10 mL to 50 mL
• Thermostatically controlled heating rod
• Stirrer to keep the water in the outer jacket at uniform temperature
• Oxygen or delivery glass tube which also serves as tissue holder
• One glass coil, one end of this is connected to the lower end of the tissue bath and other end connected to the
container/reservoir having the physiological salt solution (The glass coil is usually of double the capacity of
the inner organ bath to ensure warming up of the solution before it enters the organ bath).
o Sherrington rotating drum and Kymograph paper
o Recording levers
❖ Organ bath Assembly
 ❑ Physiological Salt Solution

➢ The physiological salt solution (PSS) was used to keep the isolated tissue or organ preparation surviving as
long as the experiment is over.
➢ PSS is very important to maintain tissue outside the animal body which fulfills their internal
environment of ions and nutrition.
➢ It is important to choose particular type of solution in which tissue is known to survive.
➢ Solution prepared with the help of distilled or double distilled or deionized water.
➢ Main components of PSS are sodium (Na+), chloride (Cl–), potassium (K+), magnesium
(Mg+), calcium (Ca+) and glucose.
➢ Aeration is important for solution with oxygen (O2) or 95% O2 + 5% CO2 (carbogen). This
process helps to provide O2 to tissue and mix PSS thoroughly in organ bath. Additionally,
carbogen is important because, pure O2 may interact with bicarbonate (HCO3–) buffer in PSS
which will cause CO2 loss and PSS become alkaline.
➢ Certain precautions must be taken, while addition of ingredients into the distilled water
(DW)/double distilled water (DDW) or deionized water (DIW).
➢ Calcium chloride should be added at last, to prevent precipitation or chelation of the bicarbonate
which make solution turbid or opaque. This may interfere with internal property of solution and
may reduce visibility of tissue in inner organ bath.
➢ Thumb rule for selection of PSS is that, Tyrode may be used for experiment with non-innervated
muscle while Krebs is used for nerve muscle preparation.
➢ The pH is important to maintain PSS property and should be at range 7.3-7.4.
➢ Fresh solution is always prepared while it can be stored for 24 h in fridge, but storage can’t be
preferred for longer time because of microbial growth (due to presence of glucose). If preparation
of solution is needed and requires storage longer than 24 h then avoid addition of calcium and
glucose which should be added at the time of experiment
 ❑ Response Recording Procedure

• Isolate the tissue and tie it in the tissue bath

 ❑ Set up the recording lever and adjust the magnification

o Levers are used to record the contractions or relaxations of the isolated tissue preparations. The
recording is done on smoked paper fixed on circular cylinder and run at different speed using
electrical recording drum.
o The writing levers are light in weight, rigid and are generally made up of wood, light aluminum
or stainless steel.
➢ Levers are of two type:
▪ Isotonic type: change in length due o contraction/relaxation is recorded while the tension on the
muscle remain the same. E.g.: simple lever, frontal writing lever.
▪ Isometric lever: isometric recording measures increases intension of the tissue when the length is
kept constant.
Simple lever: It is the simplest type of lever made up of wood, stainless steel or aluminum. A
celluloid writing tip (stylus) I attached at the end of the longer arm. The contractions are recorded as
curved lines.

Frontal writing lever(writes frontally): this lever is designed in such a way that the writing point
rotates freely about its axle. This helps in reducing the tension between smoked paper and the
recording tip. The contractions are recorded as straight line.
Starling’s heart lever: This lever is used to record the contractions of the heart. The difference
between this and other isotonic levers is that the fulcrum lies at one end beyond the point of
attachment.
❑ Magnification : Depending on the inherent contractility of the tissue preparation under study, the

magnification of the response should be adjusted in order to get a proper recording of the observed

physiological response.

➢ The tissue showing less contractility need more magnification and the reverse is true with tissue which have

higher inherent rhythmic contractility.

➢ Or example: while recording the effect on guinea pig ileum or rectus abdominus muscle it is desirable to

have 5-10 fold magnification whereas, for at uterus preparation the magnification needed is only 4-6 fold.
➢ The adjustment of magnification is dine by properly adjusting the distance between the writing tip of
lever and the fulcrum, and the distance between the point of attachment to the tissue and the fulcrum.
By adjusting relative distances desired degree of magnification is obtained.

𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒃𝒆𝒕𝒘𝒆𝒆𝒏 𝒇𝒖𝒍𝒄𝒓𝒖𝒎 𝒂𝒏𝒅 𝒘𝒓𝒊𝒕𝒊𝒏𝒈 𝒑𝒐𝒊𝒏𝒕

▪ Magnification value: 𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒃𝒆𝒕𝒘𝒆𝒆𝒏 𝒇𝒖𝒍𝒄𝒓𝒖𝒎 𝒂𝒏𝒅 𝒕𝒉𝒆 𝒑𝒐𝒊𝒏𝒕 𝒐𝒇 𝒂𝒕𝒕𝒂𝒄𝒉𝒎𝒆𝒏𝒕 𝒕𝒐 𝒕𝒉𝒆 𝒕𝒊𝒔𝒔𝒖𝒆
❑ Application of load (Tension):
The tissue preparation has to be properly relaxed without affecting the normal tone and rhythmic activity so
that efficient contractions are achieved when stimulated, and it also relaxes to its full length afterwards.
➢ This is achieved in the following way
✓ Select the proper length of longer and shorter arms depending on the magnification for the tissue which is
under study, and fix the fulcrum
✓ Balance the lever by putting the weight at the end of shorter arm and mark the point of tissue attachment
✓ At equidistance i.e. the distance between the fulcrum and the point of tissue attachment, rom the fulcrum on
the longer arm of the lever, fix the desired load required for the particular tissue
➢ The tension/load prescribed for various commonly used tissue preparations are:
✓ Guinea pig ileum (1g), Guinea pig trachea (0.2g), guinea pig vas deferens (0.5g), rabbit duodenum (1-3g),
rat uterus (1g), rat colon (0.5g), rat fundus (1g) and frog rectus abdominis (1-5g).
❑ Smoking and fixing of kymograph paper:
➢ Fix the kymograph paper (glossy surface out) tightly on the drum. Then paper should be uniformly smoked
with the black soot (smoke) of benzene or kerosene or their mixture. Uniformly smoking is essential for
proper recording.
➢ The recordings on the smoked paper are preserved by properly fixing them with help of fixing solution. The
commonly used resins to prepare the fixing solution are shellac and colophony. A saturated solution of
shellac is prepared in alcohol, and it is allowed to stand for a week. The clear supernatant is decanted and is
used for fixing the tracings/responses. The solution may be reused several times and should be kept in a well
closed bottle to prevent evaporation of the solvent.
➢ Recording can be done directly on white paper (unsmoked paper) with the help of ink-writing device (pen)
attached to the tip of the lever. Such tracing can be directly used for comparison or for calculating the
results. There is no fixing required.
❑ Contact time:

The time that is allowed for the drug to remain in contact with the tissue is called the contact time. The contact

time depends upon the type of the tissue used.

For example:

▪ A slow contracting tissue such as frog rectus abdominis preparation, the contact time allowed is 90 sec.

▪ On the other hand for guinea pig ileum the contact time is 30 sec.

▪ When the drug is in the vicinity of receptors it is called in biophase or receptor compartment.
❑ Time cycle:

➢ A fixed time cycle is used while recording any effect of the drug on isolated tissue preparation. The fixed

time cycle which comprises of starting of the drum, recording the base-line, effect of the drug (contact time)

and washout/washing period.

▪ Generally a five minute time cycle is followed, which includes: 30 sec. of base line recording, 90 sec. of

contact time (response of drug) and the subsequent three washing at interval of each minute.

▪ It is very essential to follow the fixed time cycle wile doing the bioassays o obtain uniform recordings and, it

also helps in calculating the approximate time required to complete the experiments depending on the

number of response to e recorded.