This document provides an overview of in-vitro pharmacology experiments using isolated tissues and physiological salt solutions (PSS). It defines pharmacology and drugs, describes the aims of experimental pharmacology as finding therapeutic agents, studying toxicity and mechanisms of action. It also discusses types of experiments, equipment like organ baths and levers for recording tissue responses, and PSS compositions and roles. PSS are artificial solutions that maintain isolated tissues by resembling extracellular fluid composition. Selection of the appropriate PSS depends on the tissue being studied.

1. Experiment No. 1
Introduction to in-vitro pharmacology and
physiological salt solutions (PSS)
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
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2. Overview of Discussion
 Definitions of pharmacology & drug
 Aims of experimental pharmacology
 Pre-clinical pharmacology
 Clinical pharmacology
 Types of experiments in pharmacology
 Assembly for isolated organ/ tissue related experiments
 Recording (writing) levers
 Physiological salt solution (PSS)
 Introduction
 Examples
 Composition
 Role of ingredients
 Precautions in preparation of PSS
 Selection of PSS 2

3. Definitions of pharmacology & drug
Pharmacology
The science which deals with the study of drugs.
The word ‘pharmacology’ is derived from the Greek words- Pharmakon (a
drug or poison) and logos (discourse/ study/ science).
It broadly covers the information about the history, source,
physicochemical properties, and physiological actions, mechanism of
action, absorption, distribution, metabolism, excretion and therapeutic
uses of drugs→ PK and PD profile of drug.
Drugs
The chemical substances used for the purpose of diagnosis, prevention,
relief or cure of a disease in man or animals.
The word drug derived from the French word ‘drogue’ meaning herb.
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4. Aims of experimental pharmacology
The main aims of experimental pharmacology are to-
(1) find out therapeutic agents suitable for human use,
(2) study the toxicity of a drug, and
(3) study the mechanism and site of action of drugs.
Experimental pharmacology involves the discovery of new drugs or to
study the actions of existing drugs.
Divisions of experimental pharmacology-
i) Preclinical pharmacology which involves the identification and
optimization of novel chemical lead structures and testing on animals
and animal tissues or organs for their biological action, and
ii) Clinical pharmacology where testing of drugs is done on human
volunteers and patients for assessing the pharmacokinetic, safety and
efficacy in humans.
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5. Definitions of pharmacology & drug
Pharmacology
The science which deals with the study of drugs.
The word ‘pharmacology’ is derived from the Greek words- Pharmakon (a
drug or poison) and logos (discourse/ study/ science).
It broadly covers the information about the history, source,
physicochemical properties, and physiological actions, mechanism of
action, absorption, distribution, metabolism, excretion and therapeutic
uses of drugs→ PK and PD profile of drug.
Drugs
The chemical substances used for the purpose of diagnosis, prevention,
relief or cure of a disease in man or animals.
The word drug derived from the French word ‘drogue’ meaning herb.
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6. Aims of experimental pharmacology
The main aims of experimental pharmacology are to-
(1) find out therapeutic agents suitable for human use,
(2) study the toxicity of a drug, and
(3) study the mechanism and site of action of drugs.
Experimental pharmacology involves the discovery of new drugs or to
study the actions of existing drugs.
Divisions of experimental pharmacology-
i) Preclinical pharmacology which involves the identification and
optimization of novel chemical lead structures and testing on animals
and animal tissues or organs for their biological action, and
ii) Clinical pharmacology where testing of drugs is done on human
volunteers and patients for assessing the pharmacokinetic, safety and
efficacy in humans.
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7. Types of experiments in pharmacology
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8. Assembly for isolated organ/ tissue related experiments
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Figure- Student organ bath and Sherrington’s drum revolving machine

9. Student organ bath
The tissue bath used to put the animal tissue for studying the drug actions is
called as student organ bath.
This was first designed by Rudolph Magnus in 1904.
The organ bath essentially consists of-
a) an outer jacket (water bath) made up of steel, glass or perspex,
b) the inner organ or tissue bath made up of glass with a capacity varying
from 10 to 50 ml,
c) thermostatically controlled heating rod,
d) stirrer to keep the water in the jacket at uniform temperature,
e) oxygen or delivery glass tube which also serves as tissue holder, and
f) glass coil, one end of which is connected to the lower end of the organ
bath and the other to the container having the physiological salt solution.
The glass coil is usually of double the capacity of inner organ bath to ensure
warming up of the solution before it enters the organ bath.
The student organ bath having two units of inner organ tissue bath is called
double unit organ bath. 9

10. Sherrington’s drum revolving machine
It is used to move the kymograph at a fixed speed.
The drum (with 152 mm of diameter) on which the kymograph is pasted is
fixed on the shaft of Sherrington’s drum revolving machine and this
drum revolves at a fixed speed around the shaft.
The shaft has a groove in which movable metal block is present. This
movable metal block is used to elevate or lower the position of drum.
It is elevated or lowered by rotating the screw located at the top of shaft.
The base of Sherrington’s drum revolving machine has three basal screws
meant for horizontal leveling of machine.
The gears in the basal part are for controlling the speed of rotating drum.
The clutch on the backside of machine is to start or stop the rotation of the
drum during recording of responses. Generally, rotation speed of this
machine ranges from 0.12 mm/second to 500 mm/second or more.
The strikes are used to provide electrical stimuli to the isolate tissue at a
particular frequency.
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11. Recording (writing) levers
They are used to record the contractions or relaxations of the isolated
tissue preparations. The recording is done on smoked papers fixed on
circular cylinders (of different diameters) and run at different speed
using electrical recording drums. The speed of the drum is adjusted
depending upon the nature of experiment.
The writing levers are light, rigid and are generally made up of wood
(straw), light aluminium or stainless steel. The levers are of two types-
(i) Isotonic type or type-I levers, which records the change in length due to
contraction when the tension or applied load remains constant.
Examples of isotonic levers are simple lever, frontal writing lever etc.,
and
(ii) Isometric type or type-II levers, which records increase in the tension of
the tissue due to contraction when the length of the tissue is kept
constant. These are used in special circumstances such as recording
muscle twitches produced by electrical stimulation. For recording such
observations isometric strain-gauge transducer may be preferred.
Examples of isometric levers are Starling’s heart lever, Brodie’s
universal lever, gimble lever etc.
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12. 12
Fig. Different types of recording levers

13. (a) Simple lever (side way writing) It is used to record isotonic contractions
in the isolated tissue. It is the simplest type of lever made up of wood,
stainless steel or aluminium. A celluloid writing tip (stylus) is attached at
the end of the longer arm. The contractions are recorded as curved lines.
Uncontrolled friction between stylus and kymograph is a major
disadvantage of simple lever.
(b) Frontal writing lever (writes frontally) It is used to record isotonic
contractions in the isolated tissue. This lever is designed in such a way
that the writing point rotates freely about its axle. This helps in reducing
the tension between the smoked paper/ kymograph and the recording
tip. The contractions are recorded as straight line.
(c) Starling’s heart lever It is used to record isometric contractions in the
isolated tissue. This lever is used to record rapid and multiple
contractions in the isolated tissues like frog’s isolated and perfused
heart. In this, the horizontal arm of the lever is suspended to a rigid
point with a spring. The difference between this and other isotonic
levers is that the fulcrum lies at one end beyond the point of
attachment. 13

14. (d) Brodie’s universal lever It is a general utility lever.
(e) Gimbal lever It is used to record isometric contractions in the
isolated tissue. The friction between the writing end and the
kymograph is minimum because the pressure on the stylus on the
kymograph is depends on the gravity.
(f) Paton’s auxotonic lever It is designed in such a way that the load on
the tissue goes on increasing as the tissue contacts. In short, this
type of lever is used to record change in length of tissue due to
contraction with respect to change in applied load or tension or
force of contraction.
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15. Physiological salt solution (PSS)- Introduction
The suitable solution providing the ionic requirements and nutritional
supply to the isolated tissue/ organ under study is called as physiological
salt solution (PSS).
It provides an artificial media resembling the inorganic composition of
blood plasma together with a buffer mechanism to maintain the
optimum pH and glucose to facilitate tissue metabolism.
It contain a mixture of cations, anions and glucose in distilled water.
It is also called as PSS or Ringer solution.
A solution of a salt or salts that is essentially isotonic with tissue fluids or
blood; (especially: an approximately 0.9% solution of sodium chloride)
→ Therefore also called as normal saline, OR brine (salt water), OR
saline solution, OR normal salt solution, OR physiological saline solution,
OR physiological salt solution.
It is an artificially prepared solution used to maintain tissues in viable state.
PSS can be used to keep isolated tissue alive under experimental condition.
PSS is very important to maintain tissue outside the animal body and fulfills
their internal environment of ions and nutrition.
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16. Physiological salt solution (PSS)- Examples
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17. Ringer, frog ringer and De-Jalon solution do not contain phosphate
(PO42-) ions.
The composition of De-Jalon solution is same as that of Ringer-Locke
solution except that it contains 1/4th the amount of calcium chloride
(CaCl2) and ½ the amount of glucose.
McEwen solution in addition contains sucrose.
Frog ringer solution may also be prepared by adding 400 ml distilled
water to one litre of Ringer-Locke solution (single glucose).
Ringer solution is aerated with oxygen or air and used for mammalian
isolated heart and other tissue, frog ringer is used frog heart and
other isolated tissue preparations.
Tyrode solution is aerated with air, oxygen or 5 % CO2 in oxygen and
used for mammalian smooth muscles.
Krebs and McEwen solutions are aerated with 5 % CO2 in oxygen and
used for mammalian isolated organ especially for nerve responses.
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18. Physiological salt solution (PSS)- Composition
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The main components of PSS are
Sodium
Na+
Potassium
K+
Calcium
Ca2+
Chloride
Cl- Glucose
Magnesium
Mg2+
Distilled
Water
(D. W.)
De-ionized
Water
(D. W.)
Doubled
distilled
Water
(D. W.)
Bicarbonates
HCO3-
Phosphate
Buffer

19. Physiological salt solution (PSS)- Role of Ingredients
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Sodium
Na+
Potassium
K+
Calcium
Ca2+
Magnesium
Mg2+

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Bicarbonates
HCO3-
Phosphate
Buffer
(KH2PO4/
NaH2PO4)
Glucose
Water
Energy source, provide nutrition
Vehicle/ Solvent

21. Physiological salt solution (PSS)-
Precautions in preparation of PSS
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Maintain PH = 7.3- 7.4
Hygroscopic Cacl2 & Mgcl2 salts to
be added from stock solution
Replacement of Mgcl2 with MgSO4 do not
interfere with tissue activity
Risk of bacterial growth due to glucose→ if stored for longer
duration.
CaCl2 added last → to prevent precipitate (PPT) formation or chelation
of bicarbonate. (PPT makes solution turbid It interferes with internal
property of solution & may reduce visibility of tissue).

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Add glucose & calcium at the time of Expt. if needed and
required to store for 24 hrs.
PSS should be stored for 24 hours in refrigerator
Always prepare fresh solution
Tissue aeration with the mixture of 95% O2+ 5% CO2 (Carbogen)
O2 is Important for survival of tissue.
Pure O2 interact with bicarbonate (HCO32-) in PSS causing CO2 loss
to produce alkaline solution.
Error should be less than 1%

23. Physiological salt solution (PSS)- Selection of PSS
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Tyrode solution →
For non-innervated muscles
Krebs solution →
For innervated muscles
Frog ringer solution →
For amphibian tissues
Ringer Locke solution →
For isolated amphibian heart
Tyrode solution →
Smooth muscles
Ringer Locke solution →
For isolated amphibian heart
Krebs, De Jalon’s and
Mc-Ewen solution →
For avian skeletal muscles and
other innervated muscles
De Jalon’s solution →
For isolated tissues of rabbit

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Thank you!