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Experiment No: 01
Experiment Name: Observation of the effect of water on diuresis of an adult healthy
frog.
Objective:
Diuresis: Diuresis is an increase in the production of urine by the kidneys, which
typically results in a corresponding increase in urine expelled by the body; diuresis
without an accompanying increase of urination can cause severe medical
problems. There are a wide range of causes for diuresis, and an assortment of
treatment approaches, when treatment is required.
Diuretics: Diuretics are drugs that enable the body to get rid of excess fluids, via
urination. Commonly called "water pills", some diuretics are available over-thecounter while others require a doctor's prescription.
The term "diuretic" is very broad, and refers generally to any substance that cause
the kidneys to get rid of extra water and salt. There are many kinds of diuretics,
and each diuretic works on different parts of the kidneys.
Some of the common classes of diuretics include:
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Thiazide diuretics (hydrochlorothiazide)
Loop diuretics (furosemide)
Potassium sparing diuretics (spironolactone)
Each class of diuretic has a different specific mechanism of action, but all
typically shares the ultimate outcome of changing the way the kidneys handle salt,
water, and other substances.
Common Diuretic Names:
Many different diuretics are available. Some commonly prescribed diuretics
include:



Furosemide
Hydrochlorothiazide, also known as HCTZ
Spironolactone

Metolazone
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Many other diuretics are available, both within the United States and around the
world. However, the vast majority of prescriptions in the U.S. are for the drugs
listed above. Other types of diuretics are used in special circumstances or
controlled hospital settings
Uses of Diuretics: In medicine, diuretics are used to treat heart failure, liver
cirrhosis, hypertension and certain kidney diseases. Some diuretics, such as
acetazolamide, help to make the urine more alkaline and are helpful in increasing
excretion of substances such as aspirin in cases of overdose or poisoning.
Diuretics are often abused by sufferers of eating disorders, especially bulimics, in
attempts at weight loss
The antihypertensive actions of some diuretics (thiazides and loop diuretics in
particular) are independent of their diuretic effect. That is, the reduction in blood
pressure is not due to decreased blood volume resulting from increased urine
production, but occurs through other mechanisms and at lower doses than that
required to produce diuresis. Indapamide was specifically designed with this in
mind, and has a larger therapeutic window for hypertension (without pronounced
diuresis) than most other diuretics.
Materials:
1. Purified Water
2. Subject (------ kg frog)
Procedure:
1. We take an adult healthy frog and weight it.
2. We take 2ml of purified water in a 10ml sterile syringe which has just been
opened from a sterile packaging.
3. We inject the pre-filled water into the subcutaneous of the left hind leg of the frog.
4. We wait for some time and observe the time for urination and the volume of urine
of the frog.
5. We repeat the procedure for 2.5ml, 3ml, 5ml of water injected into the frog.
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Table:
Volume of Water/ml
2.0
2.5
3.0
5.0
Time for Urination/min
Volume of Urine/ml
Observation: It was observed that with increase in volume of water injected, the time for
urination decreased proportionately and the volume of urine increased.
Conclusion:
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Experiment No: 02
Experiment Name: Observation of the effect of lidocaine as a local anaesthesia on
adult healthy mice.
Objective:
Anaesthesia: Anaesthesia is the induction of a state of unconsciousness with the absence
of pain sensation over the entire body, through the administration of anesthetic drugs. It is
used during certain medical and surgical procedures.
A drug, administered for medical or surgical purposes that induces partial or total loss of
sensation and may be topical, local, regional, or general, depending on the method of
administration and area of the body affected.
Anaesthesia means "loss of sensation". Medications that cause anaesthesia are called
anaesthetics. Anaesthetics are used for pain relief during tests or surgical operations so
that you do not feel any of the following:



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pain
touch
pressure
temperature
Anaesthetics: Anaesthetics is the science, study, and practice of anaesthesia and its
application. An anaesthetic is used to temporarily reduce or take away sensation, usually
so that otherwise painful procedures or surgery can be performed.
The different types of anaesthetic are described below.
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Local anaesthetic – used for minor procedures and tests to numb the nerves in the
area where the procedure is taking place. You will be conscious during the
procedure but you do not feel any pain.
Regional anaesthetic – used for larger or deeper operations where the nerves are
harder to reach. Local anaesthetic is injected near the nerves in order to numb a
larger area, but you remain conscious.
Epidural anaesthetic – a regional anaesthetic used to numb the lower half of your
body, which is often used for childbirth.
Spinal anaesthesia – a regional anaesthetic that is used to numb your spinal
nerves so that surgery can be carried out in this area.
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
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General anaesthetic – used for bigger operations when you need to be
unconscious. The anaesthetic stops your brain recognizing any signals from your
nerves, so you cannot feel anything.
Sedation – for painful or unpleasant procedures that are otherwise minor. Sedation
makes you feel sleepy and relaxes you both physically and mentally.
Uses of anaesthetics:
Acute pain
Acute pain can often be managed using analgesics. However, conduction
anesthesia may be preferable because of superior pain control and fewer side
effects. For purposes of pain therapy, local anesthetic drugs are often given by
repeated injection or continuous infusion through a catheter. Low doses of local
anesthetic drugs can be sufficient so that muscle weakness does not occur and
patients may be mobilized.
Some typical uses of conduction anesthesia for acute pain are:
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Labor pain (epidural anesthesia)
Postoperative pain (peripheral nerve blocks, epidural anesthesia)
Trauma (peripheral nerve blocks, intravenous regional anesthesia, epidural
anesthesia)
Chronic pain
Chronic pain of more than minor intensity is a complex and often serious
condition that requires diagnosis and treatment by an expert in pain medicine.
Local anesthetics can be applied repeatedly or continuously for prolonged periods
to relieve chronic pain, usually in combination with medication such as opioids,
NSAIDs, and anticonvulsants.
Surgery and dentistry
Virtually every part of the body can be anesthetized using conduction anesthesia.
However, only a limited number of techniques are in common clinical use.
Sometimes conduction anesthesia is combined with general anesthesia or sedation
for the patient's comfort and ease of surgery. Typical operations performed under
conduction anesthesia include:

Dentistry (surface anesthesia, infiltration anesthesia or intraligamentary
anesthesia during restorative operations or extractions, regional nerve blocks
during extractions and surgeries.)
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
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Eye surgery (surface anesthesia with topical anesthetics, retrobulbar block)
ENT operations, head and neck surgery (infiltration anesthesia, field blocks,
peripheral nerve blocks, plexus anesthesia)
Shoulder and arm surgery (plexus anesthesia, intravenous regional
anesthesia)[1]
Heart and lung surgery (epidural anesthesia combined with general anesthesia)
Abdominal surgery (epidural/spinal anesthesia, often combined with general
anesthesia)
Gynecological, obstetrical and urological operations (spinal/epidural
anesthesia)
Bone and joint surgery of the pelvis, hip and leg (spinal/epidural anesthesia,
peripheral nerve blocks, intravenous regional anesthesia)
Surgery of skin and peripheral blood vessels (topical anesthesia, field blocks,
peripheral nerve blocks, spinal/epidural anesthesia)
Other uses:
Topical anesthesia, in the form of lidocaine/prilocaine (EMLA) is most commonly
used to enable relatively painless venipuncture (blood collection) and placement of
intravenous cannulae. It may also be suitable for other kinds of punctures such as
ascites drainage and amniocentesis.
Surface anesthesia also facilitates some endoscopic procedures such as
bronchoscopy (visualization of the lower airways) or cystoscopy (visualization of
the inner surface of the bladder).
Materials:
1. Lidocaine 0.1% sterile solution (Market Name)
2. Subject (----------- kg mice)
Procedure:
1. We take an adult healthty mouse and weight it.
2. We take 2ml of Lidocaine 0.1% sterile solution in a 5ml sterile syringe which has
just been opened from a sterile packaging.
3. We inject the Lidocaine 0.1% sterile solution into the muscle of the left hind leg of
the frog.
4. We wait for some time and observe the time for onset of anaesthrsia and the
duration of anaesthesia of the frog.
5. We repeat the procedure for 2.5ml , 3ml, 5ml of Lidocaine 0.1% sterile solution
injected into the frog.
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Table:
Volume of Lidocaine
0.1% sterile solution/ml
2.0
2.5
3.0
5.0
Onset of anaesthesia/min
duration of
anaesthesia/min
Observation:
It was observed that with increase in Volume of Lidocaine 0.1% sterile solution, the
time for onset of anaesthesia decreased proportionately and the duration of
anaesthesia increased.
Conclusion:
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Experiment No: 03
Name of the Experiment: Estimation of Uric acid level in human body by
enzymatic and colorimetric method.
Objective:
Definition:Uric acid is a waste product normally present in the blood as a result of
the breakdown of purines. Excessive amounts of uric acid can cause crystals to
form in the joints and cause gout.
Uric acid is a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen
with the formula C5H4N4O3. It forms ions and salts known as urates and acid
urates such as ammonium acid urate. Uric acid is a product of the breakdown of
purine nucleotides. High blood concentrations of uric acid can lead to a type of
arthritis known as gout. The chemical is associated with other medical conditions
like ammonium acid urate kidney stones.
Effects of Uric acid in human body:
1. Uric Acid is a Byproduct of Normal Body Functions
Uric acid occurs naturally in the human body as a result of the metabolism of
purine. Purine is a muscle protein that enters the body either through dietary intake
(about 30 percent) or from the breakdown of the body's own cells during cell
turnover (about 70 percent). Normal blood serum uric acid levels are 2.4 to 6.0
mg/dl for woman and 3.4 to 7.0 mg/dl for men.
2. Humans Cannot Metabolize Uric Acid, So It Is Excreted
Humans lack the enzyme (uricase) that breaks down uric acid. So uric acid is
excreted by the kidneys and the intestines. Under normal circumstances about 70
percent of uric acid is excreted in the urine, with the intestines passing the
remaining 30 percent. However, when renal function is insufficient, a greater
percentage is excreted via the intestines. Uric acid is relatively insoluble. So, when
concentrations exceed normal levels, it may crystallize in the joints, the urinary
tract or under the skin.
3. High Uric Acid Is Associated With Kidney Problems
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Renal failure is associated with high uric acid levels, and uric acid crystals may
accumulate in the kidneys, as kidney stones, or in the bladder. But kidney
problems cannot be assumed to be caused by high uric acid. Rather, high uric acid
levels may be the result of renal insufficiency.
4. High Uric Acid is Associated with Gout
In gout, uric acid crystals deposit on cartilage, tendons and tissues surrounding the
joints. Symptoms include rapid onset of severe, burning pain; swelling, redness,
warmth and extreme skin tenderness. The big toe is the most common location for
gouty flair ups, but ankle, wrist, and knee and elbow involvement are also
reported, though less commonly. Men tend to have higher uric acid levels than
women. So, it's not surprising that men get gout more often than women.
However, gout can occur without high uric acid levels, and high uric acid does not
always cause gout. So, other causes for gout cannot be rule out. For instance,
heredity is thought to be a predisposing factor for gout.
5. Diet, Health and Heredity Affect Uric Acid Levels and Gout
A diet high in fat and muscle protein such as red meat, poultry, fish and other
seafood, is associated with high uric acid levels and also with gout. Though
causation is not firmly established, alcohol consumption, consumption of high
fructose corn syrup, obesity, starvation, excessive exercise and sleep apnea are
also correlated with gout. The common metabolic phenomenon among these is
that they all, in one way or another, cause the body to metabolize its own tissues,
thus releasing purine into the blood stream. Gout seems to occur more often within
certain racial groups, Pacific islanders and African Americans, for instance. Age
and sex also seem to make a difference, with gout afflicting men age 50 to 60 most
often.
Conclusion:This test is done to see if we have high levels of uric acid in our
blood. High levels of uric acid can cause gout or kidney disease.
Our doctor may also order this test if we have had or are about to have certain
types of chemotherapy. Rapid weight loss, which may occur with such treatments,
can increase the amount of uric acid in your blood.
Apparatus:
i.
ii.
iii.
iv.
Micropipette,
Test tubes,
Centrifuge machine,
Pipette.
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Reagent:
i.
ii.
iii.
Enzyme ket, it contains:
- phosphate buffer 50mole/L,
- 4-amino phenazole 0.3mole/L
- 3,5 dichloro, 2-hydroxy benzene sulfonic acid (DCHBS) 4 mole/L
- Enzyme uricase >200 unit,
- Enzyme peroxidase>1
- Stabilizer 10mg/dl
Standard uric acid,
Blood serum
Procedure:
i.
ii.
iii.
iv.
v.
vi.
vii.
viii.
At first blood was taken by a syringe and rinsed well with anti-coagulant in a
centrifuge tube.
The blood was then centrifuged for 15-20 minutes in a centrifuge machine to
obtain the serum sample,
Three test tubes were taken and marked as sample , standard and blank,
Then one ml of enzyme kit solution was placed in each test tubes,
Then 20µl of serum was taken in the sample test tube,
Then 20µl of standard uric acid solution was taken in the standard test tube,
Then all the solutions were taken in a dark place for above 15-20 minutes,
Finally the absorbance of these solutions were calculated at 510nm in a
colorimeter.
Calculation:
Sample
Blank
Standard
Sample
Àbsorbancer at 510nm
0.0000
0.0149
0.0111
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We know,
Absorbance of sample
Concentrat ion of sample
=
Absorbance of Standard
Concentrat ion of standard
So, Concentration of sample=
=
Absorbance r of sample
× concentration of standared
Absorbance of standard
0.0111
× B mg dl
0.0149
=5.9597
Result : The concentration of Uric ancid in Blood sample was 5.9597 mg/dl
Comments: The concentration of uric and in blood sample was 5.9597mg/dl. So it was
within normal limits.
Prcautions:
i.
ii.
iii.
iv.
v.
The blood should be taken form the humam body very carfully.
Antivoagulant should be rised very qucikly, otherwise blood would be clotted,
All test tubes should be washed with distilled water and marked sincerely,
Serum sample and standard uric acid solution should be taken with
micorpipette,
Absorbance should be measured very sincerely.