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Zoology Practical Assignment

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Acharya Sahana
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23 views10 pages

Zoology Practical Assignment

Uploaded by

Acharya Sahana
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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QUALITATIVE ANALYSIS OF NITROGENOUS WASTE-

AMMONIA, UREA, URIC ACID.

AIM: To detect the presence of nitrogenous waste products


like ammonia, urea and uric acid.
PRINCIPLE: Nitrogenous wastes are produced as a result of
protein metabolism. The amino acids undergo deamination.
The ammonia group removed is toxic and need to be
eliminated. Ammonia is eliminated as such in fresh water
forms, in amphibians and mammals ammonia is converted
to uric acid before being excreted.
To detect the presence of ammonia a direct test is conducted
using Nessler’s reagent. To test for uric acid a direct test is
conducted. There is no direct test for urea , hence urea is
broken down into ammonia and carbondioxide by using
urease powder. The ammonia released then detected using
Nesler’s reagent.
APPARATUS REQUIRED: Test tubes, test tube stands and
spirit lamp.
REAGENTS REQUIRED: Nessler’s reagent, saturated sodium
carbonate solution, folic’s uric acid reagent.

PROCEDURE
Test for ammonia:

Principle: Nessler’s Reagent is a solution containing K2HgI4


and KOH, Iodide and mercury ions react with ammonia under
alkaline conditions to produce a reddish brown complex. The
intensity of the color is the direct proportion to the ammonia
concentration.
Procedure: A direct test can be done for ammonia using
Nessler’s reagent is taken in a clean test tube and to this ½
ml of the sample is added. The formation of a brick red
precipitate indicates the presence of ammonia .
Test for uric acid :

Principle: uric acid is oxidized to allantion and carbondioxide


by a phosphotungstic acid reagent in alkaline solution.
Phosphotungstic acid is reduced in this reaction to tungsten
blue which can be measured at 710 nm for quantitative
estimation.
Procedure: 2ml of the sample is taken in a clean test tube
and a few drops of saturated sodium carbonate solution is
added followed by ½ ml of folin’s uric acid reagent. The
appearance of a blue color indicates the presence of uric
acid.
Test for urea:
As there is no direct test for urea, urea is broken down to
ammonia and carbondioxide. Urea can be broken down using
the enzyme urease in the form of urease powder.
2 ml of the sample is taken in a test tube , and it is boiled to
expel ammonia present in it . The sample is cooled to room
temperatures and urease powder is added. The test tube is
set aside for about 1 hour. After one hour the sample is
tested for the presence of ammonia by using Nessler’s
reagent
SAMPLE A B C D
ammonia + + - -

Uric acid - + - -

Urea - + ++ -
OBSERVATION:
ACTION OF SALIVARY AMYLASE UNDER OPTIMUM
CONDITIONS

Aim: To estimate the activity of amylase(quantitative


method using dinitro salicylate solution).
Principle: salivary amylase is a carbohydrase that acts on
starch and breaks it down to glucose. The activity of any
enzyme is estimated by the presence of its end product. To
estimate the activity of amylase the endproduct or glucose is
detected by using dinitro salicylate solution. The activity of an
enzyme is defined as the micromoles of product formed per
ml of enzyme solution under ideal or optimum conditions.
Apparatus required: test tubes, test tube stand , test tube
holders and water bath.
Reagents required:
Human saliva (diluted with phosphate buffer 6,9 to a make a
solution of 1:10 ). 3.5 dinitro salicylate solution,
1% starch solution,
Standard glucose solution, (1g of glucose in 100ml of water)
Distilled water.
DNS reagent-
To 100ml of distilled water add the following in a sequence-
a. Sodium hydroxide – 1gm
b. Phenol – 2ml
c. Sodium potassium tartarate – 20gm
d. Sodium carbonate - 0.05gm
e. 3,5- dinitro salicylic acid – 1gm

Procedure:
This experiment is conducted in two parts.
Part A
The first part is to get a standard graph.
Take ten clean test tubes and pipette out different aliquots of
standard glucose solution i.e, to test tube number 1 add 0.1
ml of standard glucose solution, and so on till test tube
number 10 contains 1 ml of standard glucose solution.
To each of the test tubes add the correct amount of
water to make up the volume to 2ml . Now add 2ml of DNS
solution. Mix the contents of the test tube well. Place them
in a boiling water bath for 15 minutes. Cool the test tubes.
Add 16ml of distilled water and make up the volume to 20ml.
mix the contents of the test tubes well. Read the absorbance
at 540 nm against a blank solution.
Part B
Take a clean test tube add 1ml of saliva and 1ml of 1% starch.
Keep the test tube aside in a warm place for 10 minutes. The
amount of glucose formed is determined to estimate the
activity of salivary amylase. Add 2 ml of DNS and mix the
contents of the test tube well. Keep it in a boiling water bath
for 15n minutes. Add 16ml of distilled water so that the
volume in the test tube becomes 20 ml. just like before the
absorbance is read at 540 nm. This reading is compared with
the standard graph curve to estimate the amount of glucose
formed due to action of amylase.
DIFFERENTIAL STAINING OF HUMAN BLOOD CORPUSCLES
USING LEISHMAN’S STAIN

Aim: to stain blood smear using leishman’s stain and identify


the various cell type in the blood.
Principle :
Leishman’s stain is an alkaline solution of methylene blue
that undergoes a progressive demethylation with aging or
ripening to produce a mixture of methylene blue, azures
(bright blue) and methylene violet.
It is used in microscopy for staining blood smears and to
identify and differentiate between the various WBC’s. the
strain produces a contrast between the cytoplasm and the
nucleus. The nuclear appear a brilliant violet colour and the
granules are also visible.
Requirements:
Take two slides and using a cotton swab wipe the slides with
90% alcohol and place it on a sheet of blotting paper.
Using a sterilized needle or lancet prick your finger. Wipe out
the first drop with sterilized cotton swab and then allow a
drop of blood to fall on the slide.
Allow it to dry.
Place the slide in a petri dish with the smear side up. Using a
dropper gently cover the smear with leishman’s stain. Allow
the cells to strain.
After a minute or two, using a dropper add distilled water on
the smear and wash off the excess strain in the petri dish.
( another method is to use a staining jar filled with leishman’s
stain and dip the slide in it for a minute.
After washing it with distilled water allow it to dry-
Using glycerine or an oil emulsion place a cover slip on the
smear without inclusion of air bubbles. Observe under the
microscope using 100X magnification.

Observation :

The RBC’s are enucleated. The WBC’s are nucleated and we


can differenrtiate the types by looking at the nucleus,
cytoplasmic granules and cell size.

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