ANSWERS FOR ACTIVITY 7: TEST DETERMINATIONS FOR OTHER REDUCING SUGARS

A. LACTOSE
Lactose in urine (Lactosuria)
 Lactose may appear in urine in:
 late in normal pregnancy or during lactation
 Patients who had been on a milk diet for a long time
 Small amount is normally excreted in the urine about 50mg in 24 hours
 Increased levels are seen in urine in times of lactase deficiency (Lactose intolerance: high levels of
sugars accumulate in the gut, and lactose will be absorbed and excreted in urine) and when there is
intestinal damage

Qualitative test for lactose Reagents Positive Result

A. Rubner’s Test Lead acetate Lactose will cause the formation of
Concentrated NH4OH a brick red solution and then a red
precipitate

Other results: Glucose will cause a

yellow solution and yellow
precipitate
B. Ormsby’s Test Methylamine hydrochloride An intense red color is positive for
NaOH lactose

C. Mucic Acid Test Concentrated HNO3 (nitric Fine white precipitate of mucic
acid) acid crystals indicates presence of
Water lactose or galactose

D. Phenylhydrazine/Osazone Test Phenylhydrazine Formation of lactosazone crystals

hydrochloride appearing as cottonball shaped
Sodium acetate (hedgehog)

E. Woehlk’s Test NH3 Red color

15% KOH

Lactosazone Crystals
B. FRUCTOSE
Fructose in urine (Fructosuria)
 Is a rare benign condition due to deficiency of fructokinase, blood levels become elevated after
ingestion of fructose and it appears in the urine.
 As a result of excessive fruit or honey ingestion

Qualitative tests for fructose Reagents Positive result

A. Seliwanoff’s Test Seliwanoff’s reagent Fructose will cause an intense orange red
 Hot HCl converts - Consists of: color and dark precipitate w/c dissolves
fructose to Resorcinol w/ ethanol
hydroxymethyl Concentrated HCl
furfural w/c links w/
resorcinol to form a
red colored
compound
B. Borchardt’s Test Hydrochloric acid Red precipitate after heating the urine
Resorcinol sample with HCl and resorcinol

Potassium hydroxide Yellow colored acetic ether layer after

C. Barfoed’s Test
Acetic ether reaction with potassium hydroxide and
acetic ether
Barfoed’s reagent Red precipitate of cuprous oxide
- Consists of:
Cupric acetate As a confirmatory test: Add 10 cc of
Acetic acid water and 1 cc of phosphomolybdic acid
reagent and mix.
(+) reaction: Very dark, almost opaque,
blackish hue/color will appear after
addition of phosphomolybdic acid
reagent

Seliwanoff’s test:
 Precautions necessary for a positive test for fructose:
 The concentration of the hydrochloric acid must NOT be more than 12 percent
 The reaction (red color) and the precipitate must be observed after not more than 20-30
seconds of boiling.
 Glucose must NOT be present in amounts exceeding 2 percent. Glucose in the urine may be
transformed to fructose by the catalytic action of hydrochloric acid causing a false positive
reaction.
C. PENTOSE
Pentose in urine (Pentosuria)
 Xylose, arabinose and ribose
 Excessive fruit (plum and cherries) ingestion
 Rare genetic defect due to L-xylulose reductase deficiency
Qualitative tests for pentose
A. Bial-Orcinol test
 By heating w/ HCl, pentose is
converted to furfural w/c reacts
w/ orcinol to form a colored
compound
B. Tauber’s Test
C. Tollen’s Test
D. Cole Test
E. Aniline Test
F. Phenylhydrazine/Osazone Test
ANSWERS FOR ACT. 8 KETONE BODIES
Qualitative Tests for Acetone
I. Color Reaction Tests
 Principle: Sodium nitroprusside is decomposed by acetone to ferrocyanate and ferric oxide in an
alkaline solution to produce a reddish-purplish complex
Test
1. Legal Test
2. Lange Test
3. Jackson-Taylor’s Test
4. Rothera’s Test
5. Rantzmann’s Test
***
1. Frommer’s Test
Principle: One molecule of
salicylic aldehyde combines
w/ one molecule of acetone
to form oxybenzol-acetone.
Oxybenzol-acetone in the
presence of strong alkali
forms dioxy-dibenzol acetone
Reagents Positive result
Bial orcinol reagent Olive-green solution in amyl alcohol
- Consists of
Orcinol
Concentrated HCl
10% FeCl3
Tauber’s reagent Pink to red color (cherry red)
- Consists of
Benzidine
Glacial acetic acid
HCl and phloroglucinol Cherry red color
Blood charcoal Green color
Glacial acetic acid Bright red color soluble in chloroform
Pure aniline
Phenylhydrazine HCl Pentosazone crystals w/c appear
Sodium acetate as mass of entangled rootlets
Reagents Positive Result
NaOH or KOH Purple or violet-red colour
Sodium nitroprusside * Alcohol or acetic aldehyde and
Concentrated acetic acid diacetic acid give a red colour
Glacial acetic acid Purple or purplish red ring
Saturated solution of sodium * Diacetic acid gives the same
nitroprusside reaction
28% NH4OH
Sodium nitroprusside Reddish-purple ring
Ammonium sulfate powder
NH4OH
Ammonium sulfate powder Red to purple ring
Sodium nitroprusside * A pale purple ring is negative
Strong ammonia water * Diacetic acid gives a similar reaction
Rantzmann’s reagent Purple ring
28% NH4OH
Potassium hydroxide Intense red purple ring
powder
10% alcoholic solution of
salicylic aldehyde
 responsible for the intense red
color.

II. Diffusion test for acetone

 Procedure:
 Place 1 mL of Nessler’s reagent in a small watch glass resting in a petri dish.
 Acidify 2 mL of urine with a drop of glacial acetic acid or hydrochloric acid.
 Carefully pour the urine into the petri dish around the watch glass, taking care not to mix the
liquids.
 Replace the cover on the petri dish.
 Principle:
 Acetone in the urine being volatile diffuses and is fixed inside the covered petri dish which would
react with the Nessler’s reagent producing rapidly a cream coloured precipitate.

III. Iodoform tests

Test
1. Lieben’s Iodoform Test
2. Gunning’s Test
Reagents Positive Result
Strong KOH solution a. Characteristic antiseptic odour of
Iodine iodoform
Potassium iodide b. Microscopic appearance of yellow
thin hexagonal or six sided plates or
six-pointed stars of iodoform crystals
Alcoholic iodine solution The liquid turns black at first due to
Ammonia formation of nitrogen iodide, iodoform
KOH crystals are deposited and characterized
Potassium iodide by an antiseptic odour and microscopic
appearance (yellow thin hexagonal or
six sided plates or six pointed stars)

IV. Turbidity Test

Test Reagents
1. Wallhausser’s Test Scott-Wilson’s reagent
composed of mercuric
cyanide, silver nitrate, and
NaOH
Positive Result
Urine turns turbid w/ the addition of a
drop of Scott-Wilson’s reagent

Qualitative Tests for Diacetic acid or Acetoacetic acid

Test Reagents Positive Result
1. Gerhardt’s Test 10% aqueous solution of “Bordeaux red” or Wine red”
Principle: Ferric ions chelate w/ FeCl3
enol groups of diacetic acid to False (+) results: A wine red colour is
produce a “Bordeaux red” or also produced by salicylates, phenols,
“wine red” complex various antipyretics, sodium
bicarbonate,

Confirmatory procedure:
Boil the preparation showing a
Bordeaux or wine red colour

Wine red colour produced by

interfering substances would remain
after boiling

Wine red colour produced by

diacetic acid disappears after boiling.
During boiling, diacetic acid complex
is oxidized to acetone w/c is volatile
 and w/c does not form a coloured
complex with FeCl3.
2. Lindemann’s Test 30% Acetic acid + reaction: Chloroform will not change
Iodine solution colour (colourless)
Chloroform - reaction: Chloroform becomes
reddish-violet in colour
3. Arnold’s Test 1% sodium nitrate Violet purplish colour
Acetophenone solution Specific for diacetic acid
Ammonia
Concentrated HCl
4. Legal Test NaOH or KOH Purple or violet-red colour
Sodium nitroprusside
Concentrated acetic acid
5. Lange Test Glacial acetic acid Purple or purplish red ring
Saturated solution of
sodium nitroprusside
28% NH4OH
6. Rothera’s Test Ammonium sulfate Red to purple ring
powder
Sodium nitroprusside
Strong ammonia water

Qualitative Tests for β-hydroxybutyric acid

Test Reagents Positive Result
1. Hart’s Test Acetic acid Tube w/ hydrogen peroxide: red ring at
Principle: is an indirect method Distilled water the point of contact
for the detection of β- Hydrogen peroxide Tube w/o hydrogen peroxide: no
hydroxybutyric acid. The first Reagents for Legal test change in reaction, there is absence of
part of the procedure used procedure or any red ring (negative control)
boiling to break down the sodium nitroprusside
diacetic acid when present in procedures.
urine to acetone. Acetone is
removed by evaporation.
Next, the β-hydroxybutyric acid
is oxidized to diacetic acid and
acetone by the use of
peroxide. The diacetic acid
and acetone produced can
be detected by any of the
nitroprusside procedures.

2. Osterberg Test Concentrated NH4OH If the concentration of β-hydroxybutyric

Sodium nitroprusside acid is greater than 0.5% :purple color
Distilled water obtained is deeper in shade than the
standard
If the concentration is less than 0.5%:
Purple color obtained is lighter than the
standard
ACTIVITY 9: BLOOD
Qualitative Tests for Blood
Test
1. Benzidine Test
Principle: The peroxidase-like
activity of haemoglobin
decomposes hydrogen
peroxide and the liberated
oxygen oxidized the benzidine
base forming a coloured
derivative (faint green to deep
blue)
2. Guaiac Test
3. Ortho-tolidine Test
4. Teichmann’s test
5. Phenolphthalein Test
6. Pyramidone ring Test
ACTIVITY 10: BILE PIGMENTS (BILIRUBIN)
Qualitative Tests for Bile pigments (bilirubin)
Principle: Most of the tests for bile pigments are based on their precipitation by certain chemicals followed by
their oxidation to various coloured compounds such as biliverdin (green), bilicyanin (blue), biliprasin (violet),
choletelin (yellow) and other derivatives, w/c cause the appearance of bands of rainbow colours at the
junction of the oxidizing agent and the pigments present in the urine sample.
Test
1. Rosenbach’s Modification
of Gmelin’s Test
2. Harrison Spot Test
Principle: Barium chloride
combines with sulfate radicals
in the urine forming a
precipitate of barium sulfate.
Any bile pigments present will
adhere to these large
molecules. Ferric choride in the
presence of tricholoroacetic
Reagents Positive Result
Benzidine solution Trace: faint green
3% Hydrogen peroxide (+): green
(++): greenish blue
(+++): blue
(++++): deep blue
Blue or green colour should develop
within 10 minutes
Colour developing after 10 minutes is
not a positive result, it is due to the
oxidation of Benzidine by air.
Benzidine solutions are unstable and
changed readily when exposed to
light.
Glacial acetic acid Green to blue ring at the zone of
3% Hydrogen peroxide contact between the guaiac and
Gum guaiac ether
Ether
Ortho-tolidine reagent Greenish blue to deep blue color
Acid-peroxide
NSS Dark brown rhombic hemine crystals
Glacial acetic acid
Kastle-Meyer reagent Pink colour
3% hydrogen peroxide
33% acetic acid Lilac or mauve ring
5% pyramidone in absolute
alcohol
3% hydrogen peroxide
Reagents Positive Result
Nitric acid Play of colours-green towards the urine
layer
10% Barium chloride Blue to green colour on the filter paper
Fouchet’s reagent is
composed of FeCl3 and
TCA (tricholroacetic acid)
 acid will then oxidize yellow
bilirubin to green biliverdin.
3. Smith’s iodine Test
4. Ultzmann’s Test
5. Maher’s Test
6. Huppert-Cole Test
7. Franklin Test
8. Ferric Chloride Test
9. Rosin’s Test
10. Methylene blue Test
11. Foam Test
Principle: Bilirubin alters the
surface tension of urine and
foam will develop after
vigorous shaking.
12. Van den Bergh Test
ACTIVITY 11: UROBILINOGEN
Qualitative Tests for Urobilinogen
Test
1. Ehrlich’s Test
2. Wallace-Diamond Test
ACTIVITY 12: UROBILIN
Qualitative Test for Urobilin
Test
1. Schlesinger’s Test
Principle: The reaction
between urobilin and iodine
(Lugol’s) results to a
compound w/c gives a green
fluorescence to an alcoholic
solution of zinc acetate. Bile
pigments interfere and must be
removed prior to test.
Alcoholic solution of Emerald green ring will form
iodine
25% KOH Green colour will develop
Pure HCl
20% solution of para- Green colour will develop
toluene sulfonic acid or
10% HCl
0.1% sodium nitrate
Calcium hydroxide (lime) Green colour will develop
Concentrated HCl
Alcohol
Fouchet’s and Green colour will develop w/c is
Obermayer’s reagents insoluble in chloroform
Barium chloride Green or blue colour will develop
Acetic acid
Ferric chloride
1% alcoholic iodine Green ring
solution
0.2% aqueous solution of Bilirubin is present if more than 4 drops
methylene blue of Methylene Blue were added to
obtain a blue colour
Yellow foam or Greenish-yellow foam
Diazotized sulfanilic acid Red colour
Reagents Positive Result
Ehrlich’s reagent Both fluid and foam assume red colour
NH4OH
Ehrlich’s aldehyde reagent Rose red colour
Reagents Positive Result
Lugol’s Iodine Greenish fluorescence
Schlesinger’s reagent is Fluorescence is more prominent under
composed of alcoholic UV light than under day light
solution of zinc acetate Fluorescence disappears upon
acidification
ACTIVITY 13: BILE ACIDS
Qualitative Tests for Bile Acids

Test Reagents Positive Result

1. Hay Test Sulfur powder 0.01% or more: Sulfur powder sinks
Principle: Bile acids have the at once
property of reducing the surface 0.0025% or more: Sulfur powder
tension of the fluids in w/c they sinks after gentle agitation
are contained Absence of bile acids: Sulfur
powder remains floating even
after agitation
2. Oliver Test Peptone-salicylic acid-acetic Milky Turbidity
Principle: A precipitate forms acid reagent
upon mixing of peptone and
bile acids
3. Pettenkofer Test Sucrose solution Red ring
Concentrated H2SO4