GLUCOSE, OTHER REDUCING SUGARS AND KETONES
• TESTS FOR GLUCOSE IN URINE
Test Reagent
Reagent Strip Tests Glucose reagent strip
(impregnated with glucose
oxidase in its reagent pad)
Copper Reduction Benedict’s reagent: copper
Tests (Benedict’s sulfate, sodium carbonate,
Test) sodium citrate buffer
Benedict’s Test Clinitest reagent tablet: copper
(tablet version) sulfate, citric acid, NaOH,
sodium carbonate
Method/ Principle Interpretation
• Plus system (1+, 2+, 3+, 4+) with quantitative
concentrations in mg/dL or g/dL.
• Blue to green to brown
•
• Double sequential enzyme reaction and detect only glucose • Sensitivities:
-Chemstrip: 40mg/dl
• Chromogen: potassium iodide (blue to green) → oxidized chromogen: iodide
-Multistix:75-125mg/dl
(brown)
-vChem: 45mg/dl
• Affected by: ↑SG, ↓urine temp, ketonuria (≥40mg/dl) & ↑pH= ↓sensitivity
• False positive: strong oxidizing rgts
• False negative: ascorbic acid (≥50mg/dl): AA will be oxidized by H. peroxidase
(KI won’t be oxidized) → no color form’n
• Blue(-) to green to orange(+)
• Reducing substances (Glucose, Fructose, Galactose, Lactose, Maltose,
Pentose, Ribose, Arabinose) convert cupric sulfate to cuprous oxide (in alk envi
+ heat)→ color change (blue to green to orange)
• False positive: ascorbic acid, tetracycline, salicylates, penicillin, cysteine,
homogenistic acid, glucoronates
• False negative: radiographic contrast media, sulfonamides
• Sensitivity: 200mg/dl
• Water+urine→ tablet is dissolved by sodium carbonate and citric acid
(effervescent).
• NaOH: provides alkaline medium for the reaction. • The test is reported as negative, 1/4 % (or
• Rxn of NaOH+water+citric acid:.provides heat required trace), 1/2% (+), 3/4% (++), 1% (+++), or 2%
(++++).
• Reducing substances in the urine react with copper sulfate to reduce the cupric
• any color change that occurs after 15
ions to cuprous oxide
seconds is ignored
Method:
Pass-through phenomenon
• 5 drops of urine into a glass test tube (or 0.3 mL)
• Observation: ↑ conc. of reducing
• Add 10 drops of water (or 0.6 mL) and shake
substances→ color of the mixture “passing
• Drop 1 tablet in the tube and observe the complete reaction. Do not shake yet.
through” all colors possible. (Becomes
• After the 15-second waiting period, shake the tube gently and then compare with orange [highest conc.] but then proceeds to
the color chart that is provided. change back to green-brown [low conc.])
• False positive: nalidixic acid, cephalosporins, probenecid, ascorbic acid & urinary • Mechanism: reoxidation of resultant cuprous
preservatives (formalin, formaldehyde) oxide to cupric oxide and other cupric
• False-negative: no FN if procedure is done properly complexes (green).

**To determine whether a positive copper reduction test is due to the presence of glucose or another reducing substance, both the glucose oxidase test and the reduction test must be performed and
a correlation made of the results.
**These tests depend on the ability of glucose & other reducing sugars to reduce alkaline copper sulfate reagent from cupric hydroxide to cuprous oxide.
Test Reagent
Fehling’s Test • Fehling’s A: Cupric
Sulfate in distilled water
• Fehling’s B: Rochelle
salt (Sodium potassium
tartrate), potassium
hydroxide, distilled water
Haine’s Test • Haine’s Reagent:
Copper sulfate, NaOH,
Glycerol, Distilled water
Trommer’s 10 % KOH, 10 % copper
sulfate
Nylander’s Nylander’s Reagent:
Rochelle salts (Na or K
lactate), 10 % NaOH or KOH;
bismuth subnitrate
**addt’l: Two drop method • If “passthrough” occurs (bright orange to dark
• Add 2 drops urine to 10 drops water (special color chart is used) brown or greenish-brown) report as greater
• Allow quantitation upto 5% (“pass-through” may still occur at very large conc. of than 2%.
sugar) • If measurement beyond 2% is medically
desirable: do two-drop method
• Two drop method results:
OTHER QUALITATIVE TESTS
Principle Interpretation
• Glucose and other reducing sugars reduce alkaline copper sulfate reagent from • (+): Yellow precipitate
cupric hydroxide to cuprous oxide.
• Glucose and other reducing sugars reduce alkaline copper sulfate reagent from • (+): Yellow to red precipitate
cupric hydroxide to cuprous oxide.
Method:
• 4 ml Haine’s Solution is boiled and 6-8 drops of urine is added while keeping the
reagent hot but not boiling.
• (+): Precipitate of yellow cuprous hydroxide
or red precipitate.
• Glucose and other reducing sugars reduce alkaline copper sulfate reagent from
cupric hydroxide to cuprous oxide.
• If (+) result spreads out the entire field → red sediment is formed. (If this takes
place upon heating, repeat test, but omit heat)
• Reduction of bismuth to metallic bismuth in a hot alkaline solution. • Black color: Positive (+)
• Brown color: Positive (Trace)
• White precipitate: Negative (Phosphates
may be present)
 **Albumin in large amount may give similar
reaction but may be removed by boiling and
filtration

Moore-Heller’s 10 % KOH • Caramelization of sugars by strong alkali and heat. Result interpretation are as follows:
• 1 % or less- canary yellow
• 1-2 %- wine yellow
• 2-3 %- cherry yellow
• 3-4%- rum color
• >4 %- Dark brown or black color

Phenylhydrazine Phenylhydrazine and sodium • Each sugar forms a definite crystalline form when it come contact with • (+): phenylglucosazone crystals (fine bright
(Osazone) acetate phenylhydrazine and acetate (crystals separate when sugar is present). yellow needles arranged in bundles, sheaves
or rosettes).

Kowarsky’s Pure phenylhydrazine, glacial • (+) result: phenylglucosazone crystals (fine

Phynyhydrazine acetic acid and saturated bright yellow needles arranged in bundles,
(Blumel’s solution of NaCl sheaves or rosettes).
modification)
Glucose-Oxidase o-toluidine • Uses o-toluidine as indicator reagent • (+) result: Purple color in the test area.
Paper strip test • (-) result: test area remains red
**could be false neg. in presence of ascorbic
acid.

**Depends on glucose and other reducing substances to reduce the copper sulfate to cuprous oxide and in the presence of potassium thiocyanate is changed to white copper thiocyanate. Endpoint:
when all blue color is gone and only a gray color remnant
Test Reagent
Benedict’s test • Copper sulfate (pure crystallized):
Main reagent
• Sodium carbonate: provides
alkaline medium
• Trisodium citrate: Prevents
precipitation of cupric ions as
cuprous carbonate or cuprous
hydroxide by forming a loosely
bound complex with copper
(trisodium citrate: cupric complex)
which on dissociation gives
continuous supply of copper ions.
• Potassium sulfocyanate:
Precipitates cuprous ions as
cuprous cyanide (white colored
precipitate)
• Potassium ferrocyanide: Prevents
precipitation of cuprous oxide
• Distilled water
Fehling’s test
Robert
Fermentation test
Somogyi method
OTHER QUANTITATIVE TESTS
Method/Principle Interpretation
Method: Normal values:
1) Pipette 5 ml of Benedict’s Quantitative reagent into a 16 x 100 test
tube
2) Add 1- 2 grams of Anhydrous Sodium Carbonate and boil vigorously
while agitating the tube.
3) Using 1 ml serologic pipet calibrated 0.01, add the urine drop by
drop, keeping the solution boiling. When blue color starts to fade, the
solution should be boiled for 30 seconds between addition of each
drop.
4) End point is reached when blue color just disappears and a gray
color remains
**If less than 1 ml of urine is used up to the end point, make a 1:5 dilution
with dH2O and repeat procedure. In the computation, multiply the number
of ml used up to the end point by the dilution factor.
5) Record the amount of urine used up to the end point and compute
using the following formula:
Method
• 1 ml of Fehling’s solution + 4 ml of distilled water→ heated to boiling.
• Urine is added drop by drop until the grayish white color is obtained.
 TESTS FOR OTHER REDUCING SUGARS
FRUCTOSE
Test Reagent Method/Principle Interpretation
• Seliwannof’s Reagent: combination • Hot HCl converts fructose to hydroxymethyl furfural (HF links with • ( + ) result: intense orange – red color
Seliwannof’s test of resorcinol and concentrated HCl. resorcinol to form a red colored compound) and dark precipitate which dissolves with
• Concentrated HCl must not be more • Reaction must be observed NOT after more than 20-30s of ethanol
than 12 % boiling • ( - ) result: absence of red orange color
• False positive: if glucose >2% ( glucose converts to fructose by and dark precipitate
HCl)

Method:
1. Pipette 3 ml of Selivanoff’s reagent into 13x100 mm test tube.
2. Add 6 mL of urine sample
3. Heat the preparation to boiling.

• Borchardt’s reagent: combination of • Same principle with Seliwannof’s • (+) result: red precipitate after heating
Borchardt’s Test resorcinol and concentrated HCl with the urine sample with HCl and resorcinol
addition of potassium hydroxide and and a yellow-colored acetic ether layer
acetic ether. after reaction with KOH and acetic ether.

• Barfoed’s reagent: cupric acetate, • Cupric acetate is reduced from cupric hydroxide to cuprous oxide • (+) result: red precipitate of cuprous
Barfoed’s 38% acetic acid, & distilled water. by the action of fructose. oxide.
• Confirmatory test: add of 10 ml water + 1 ml of • Confirmatory test (+) result: very dark,
phosphomolybdic acid. almost opaque, blackish hue /color.

LACTOSE
Test Reagent Method/Principle Interpretation
Rubner’s Lead acetate, concentrated NH4OH • (+) result for lactose: Brick red solution with cherry red or copper colored precipitate
• (+) result for glucose: yellow solution with yellow precipitate
Ormsby’s test Methylamine hydrochloride, NaOH • (+) result: intense red color

Mucic acid test Concenctrated HNO3 • (+) result: fine white precipitate of mucic acid crystals (also indicates increased galactose in
urine)

Phenylhydrazine/Osazone Phenylhydrazine hydrochloride, sodium • (+) result: form’n of lactosazone crystals appearing as cotton-ball shaped (hedgehog).
test acetate • (same + result with galactose)

Woehlk’s test NH3, 15% KOH • (+) result: red color

 PENTOSE (Xylose, arabinose and ribose)
Test Reagent Method/Principle Interpretation
Bial-Orcinol Bial Orcinol reagent: Orcinol, By heating with HCl, pentose is • (+) result: olive green solution in amyl alcohol
concentrated HCl and 10 % FeCl3 converted to furfural which reacts with
orcinol to form a colored compound

Tauber’s Test Tauber’s reagent: Benzidine, glacial acetic • (+) result: pink to red (cherry red); disregard any yellowish to brown
acid color

Tollen’s test HCl and phloroglucinol • (+) result: cherry red color

Cole’s test Blood charcoal Charcoal removes all reducing • (+) result: green color
substances except dextrose, levulose,
pentose and formaldehyde
Aniline test Glacial acetic acid, pure aniline, chloroform • (+) result: bright red color in chloroform layer
**Glucose gives an interfering green color

Phenylhydrazine/Osazone Phenylhydrazine HCl, • (+) result: pentosazone crystals (appears as mass of entangled
test Sodium acetate rootlets)

• TESTS FOR KETONES

Test Reagent Method/ Principle Interpretation

Reagent Strip Test Ketone reagent strip
impregnated with sodium
nitroprusside
(nitroferricyanide)
• (+) result: purple
• In an alkaline medium: acetoacetate reacts with nitroprusside → color beige to
purple.
• Glycine in rgt pad: enables detection of acetone.
• Sensitivities:
-Multistix: 5-10mg/dl
-Chemstrip & vChem: 50-70mg/dl
• False positive:
-sulfhydryl containing compounds (2- mercaptoethane sulfonic acid, captopril,
cysteine)
-levodopa metabolites
-phenylketones (cause red-orange coloration)
-phthaleins (bromsulphthalein, phenolsulfonphthalein red dyes)
 - High specific gravity & low pH
• False negative:
-rapid volatilization of acetone @ RT
-breakdown of acetoacetate by bacteria
-deterioration of the nitroprusside rgt in the reaction pads from exposure to
moisture, heat, or light.

Nitroprusside Acetest tablet: sodium
Tablet Test for nitroprusside, glycine, a strong
Ketones (Acetest) alkaline buffer (disodium
phosphate), and lactose.
**Lactose: helps enhance
color.
• Diacetic acid & acetone react with sodium nitroprusside and glycine in an Reporting of results (diacetic acid):
alkaline medium → purple color. • For urine:
• Acetest will not react with beta-hydroxybutyric acid -Small color block: 5–10 mg/dL of
• Sensitivity in urine: -Moderate block: 30–40 mg/dL
-Diacetic acid: 5–10 mg/dL -Large block: 80–100 mg/dL.
-Acetone: 20–25 mg/dL • For serum, plasma, and whole blood,
-Sensitivity: 10 mg/ 100 mL.
Method: -Purple color (any pink, tan or yellow
1. Place the tablet on a piece of clean, dry white paper. coloration should be ignored)
2. Put one drop of urine, serum, plasma, or whole blood directly on the tablet.
3. For urine: compare the color of the tablet with the color chart at 30 seconds. **NOTE: Substances which interfere with the
For serum/ plasma: compare the color after two (2) minutes. dipsticks will also interfere with the Acetest tablet
For whole blood: remove the clotted blood from the tablet after 10 minutes because the same reaction is involved.
and compare the color of the tablet with the chart.
4. Results are reported as “small, moderate, or large.

OTHER SPECIFIC TESTS FOR KETONES

ACETONE
Test Reagent Method/ Principle Interpretation
Rothera’s Test ammonium sulfate and sodium • Decomposition of sodium nitroprusside or sodium ferrocyanide to sodium • (+) result: red to purple ring at the zone of
(Also for Diacetic nitroprusside ferrocyanide and ferric oxide in an alkaline solution contact
acid) • These nitroprusside reaction-based tests are 15-20 times more sensitive to • (-) result: purple ring
acetoacetate than they are to acetone & do not react with beta-hydroxybutyrate.
 Method:
• Five (5) ml of urine is added with ammonium sulfate and sodium nitroprusside
and strong ammonia water is overlaid.

Lieben’s Iodoform KOH, iodine and potassium-
test iodine solution
Gunning’s Test
(Modification of
Lieben Test)
Frommer’s Test KOH and 10 % alcoholic
solution of salicylaldehyde
Lange Test Glacial acetic acid, saturated
solution of sodium
nitroprusside, 28 %
ammonium hydroxide
• False positive: occur after a heavy meal (color persist for 30 seconds then
disappears or fade within 3-4 mins). In the same way, amorphous urate gives a
brown or orange color.
• KOH, iodine and potassium-iodine solution are added to urine • (+) result: yellow precipitate of iodoform is a
positive result.
• Microscopically: thin, yellow hexagonal plates
or starlike groups of crystals.
• Same with the Lieben test EXCEPT that it involves addt’n of excess alcoholic • (+) result: Iodoform precipitate is formed
iodine solution and ammonia water. when acetone is present.
**black coloration: form’n of nitrogen iodide
but disappears.
• 1 molecule of salicylic aldehyde + 1 molecule of acetone→ form oxybenzol • (+) result: red-purple color (usually intense
acetone (in the presence of strong alkali) → forms dioxy-dibenzol acetone purplish red ring)
forming a red-purple color (usually intense purplish red ring).
• Glacial acetic acid and saturated solution of sodium nitroprusside is added with 5 • (+) result: purple/purplish red ring at the
ml of urine. Then 28 % ammonium hydroxide is overlayed→ purple/purplish red junction of the two liquids.
ring at the junction of the two liquids.

Legal’s Test • Sodium nitroprusside or sodium ferrocyanide is decomposed to sodium • (+) result: purple or violet-red color
ferrocyanide and ferric oxide in an alkaline solution, producing a purple color. **alcohol or acetic aldehyde and diacetic acid
Method: gives the same reaction
1. Place a few ml of freshly voided urine sample in 16x150 test tube.
2. Add enough NAOH or KOH to render the sample alkaline. Test with litmus
paper after each addition of alkali.
3. Add a few drops of fresh Sodium Nitroprusside solution.
4. Add a few drops of concentrated acetic acid

Jackson-Taylor’s Method: • (+) result: few purple-red color or

Test (Modification 1. Few drops of sodium nitroprusside are added in a few ml of urine. permanganate colored ring at the point of
of Legal’s Test) 2. Ammonium sulfate is added followed by ammonium hydroxide. contact indicates the presence of acetone

Diffusion test for Method: • (+) result: cream- colored precipitate.

Acetone • 1 ml of Nessler’s reagent in a small petri dish is acidified with glacial acetic acid
or HCl.
 • Acetone in the urine being volatile diffuses (fixed inside the covered petri dish)
→ reacts with the Nessler reagent→ producing a cream- colored precipitate.
Wallhausser’s Test Urine turns turbid with the addition of a drop of Scott-Wilson Reagent (mercuric • (+) result: turbid
cyanide, silver nitrate, and NaOH).
DIACETIC ACID
Test Reagent Method/ Principle Interpretation
Gerhardt’s Test • Ferric ions chelate with enol groups of diacetic acid to produce a bordeaux red or • (+) result: bordeaux red color indicated
(refer to Manual for wine-red complex. presence of diacetic acid and or interfering
the procedure • False positive: aspirin (salicylates), phenol, antipyrine and sodium bicarbonate. substances like drugs

Method:
1. Place a few ml of freshly voided urine sample in 16x150 test tube.
2. Using a pipette, add 10% Ferric chloride drop by drop until no further
precipitation occurs.

Lindemann’s Test 30 % acetic acid, iodine and
chloroform
Arnold’s Test 1% sodium nitrate,
acetophenone sol’n, ammonia
and concentrated HCl.
Confirmatory Test:
• Heat the preparation. If the Bordeaux red color DOES NOT disappear it is due to
drugs BUT if the color will disappear and will never reappear upon cooling, it is
due to diacetic acid.
• Chloroform will become reddish violet if diacetic is not present. • (+) result:
• (+) result: In the presence of diacetic acid will
produce a violet color.

BETA-HYDROXYBUTYRIC ACID
Test Reagent Method/ Principle Interpretation
Hart’s Test (refer to • Boiling breaks down diacetic acid to acetone→ acetone is removed by • Tube w/ hydrogen peroxide – red ring at the point of
Manual for the evaporation→ addition of hydrogen peroxide oxidizes remaining acetone and contact indicates presence of beta-hydroxybutyric acid.
procedure) diacetic acid. • Tube w/o hydrogen peroxide – no change in reactions

Method:
1. Place 5 ml of freshly voided urine sample in 16x150 test tube.
2. Add equal volume of water and a few drops of acetic acid.
3. Boil the preparation ½ its original volume.
4. Dilute to 10 ml with distilled water.
5. Mix and divide the contents equally into two portions. Label them I and II.
6. To tube I, add 0.5 ml hydrogen peroxide. NO addition of hydrogen
peroxide to tube II.
7. Warm both tubes gently and allow to cool.
8. Apply Legal’s test to both tubes
9. Stand for few hours.
 Osterberg test • Concentrated • (+) result:
NH4OH, sodium -Conc. of beta-hydroxybutyric acid is > 0.5%: purple
nitroprusside color is deeper in shade than the standard
and distilled - if < 0. 5%: color is lighter.
water

• TESTS FOR BLOOD

Test Reagent Method/ Principle Interpretation

Reagent Strip Reagent pad is
Method for Blood impregnated with
tetramethylbenzidine • The pseudoperoxidase activity of the heme moiety causes the reduction of
(chromogen) and peroxide and oxidation of the chromogen→ color change yellow to green.
peroxide. • Multistix uses diisopropylbenzene dihydroperoxide tetramethylbenzidine • Homogenous color change: from hemoglobin
while Chemstrip uses dimethyldihydroperoxyhexane tetramethylbenzidine. • Mottled/speckled pattern: when intact red cells lyse
• Sensitivities: and release their hemoglobin.
-Multistix: detect 6-20 RBCs/uL and 0.02-0.06 mg/dL of hemoglobin
-Chemstrip: detect 5-10 RBCs/uL and 0.02-0.03 mg/dL hemoglobin.
• False positive: strong oxidizing agents, bacterial peroxidases and blood
contamination (hemorrhoidal or menstrual blood).
- Strong oxidizing agents (ex. Sodium hypochlorite) oxidizes the chromogen
instead of the heme moiety.
- Bacterial peroxidases produced by organisms such as E.coli catalyzes the
reaction of the reagent and chromogen despite the absence of the
pseudoperoxidase activity of heme.
• False-negative: technical errors, presence of ascorbic acid (>25 mg/dL),
strong reducing substances, high specific gravity and increased nitrite (>10
mg/dL).
- Failure to mix the sample prior to dipping the rgt strip: may not detect
the intact cells (tend to settle at the bottom)
- Ascorbic acid and other reducing substances: react and consume the
peroxidase in the reagent pad.
- High SG: RCs crenate and resist lysis (necessary to detect heme)

OTHER QUALITATIVE TEST FOR BLOOD

Test Reagent Method/ Principle Interpretation
Salt Precipitation • Purpose: Differentiate hemoglobinuria from myoglobinuria Result:
Method • Principle: Larger hemoglobin molecules are precipitated by ammonium • Myoglobin present: supernatant= red; blood
(Blondheim Test) sulfate and myoglobin remains in the supernatant. reagent strip= positive.
• Procedure: • Hemoglobin present: red precipitate; supernatant=
a. Add 2.8g of ammonium sulfate to 5 mL of centrifuged urine negative blood reagent strip.
b. Mix and stand for 5 minutes
c. The urine is now 80% saturated with ammonium sulfate (optimal for hgb
precipitation).

Benzidine Test Benzidine powder, Glacial
acetic acid, 3% Hydrogen
Peroxide
Guaiac Test Guaiac gum solution,
Glacial acetic acid, 3%
Hydrogen Peroxide
Occult Tablet o-toluidine, Strontium
Test peroxide, Calcium acetate,
Tartaric acid, Sodium
bicarbonate, Red dye
• TESTS FOR BILIRUBIN
Test Reagent
Reagent strip Reagents impregnated in
the pad:
• Multistix: 2,4-
dichloroanilinediazonium
salt
• Chemstrip: 2,6-
dichlorobenzene
diazonium salt
d. Filter or centrifuge urine
e. Test supernatant with reagent strip
• Principle: Pseudoperoxidase activity of hgb or myb→ hydrogen peroxide is (+) result: green to blue color
decomposed to water and oxygen. The liberated oxygen oxidizes benzidine
to a green or blue color
• Principle: Pseudoperoxidase activity of hgb or myb→ hydrogen peroxide is (+) result: green to blue color at the zone of contact
decomposed to water and oxygen. The liberated oxygen oxidizes the
phenols present in guaiac to quinones.
Principle: (+) results: blue color within 2 minutes
• Sodium bicarbonate reacts with tartaric acid→ liberate carbon dioxide
(causes effervescence to facilitate the rxn).
• Tartaric acid & calcium acetate→ w/ strontium peroxide= form hydrogen
peroxide.
• In the presence of the pseudoperoxidase activity of hgb or myb→ hydrogen
peroxide is decomposed to water and oxygen.
• Liberated oxygen oxidizes o-toluidine to a blue color + red dye (to mask
discoloration of tablet by blood).
Method/ Principle Interpretation
• Sensitivites:
-Multistix: 0.4-0.8mg/dl
-Chemstrip: 0.5mg/dl
• False-positive: drugs or highly pigmented substances.
- Chlorpromazine and iodine metabolites (react directly w/ diazonium
salt).
- Phenazopyridine (drug) and indican (in intestinal disorders) results to a
highly pigmented urine.
• False-negative: improper specimen storage, ascorbic acid (>25 mg/dL)
and high concentrations of nitrite.
-Must be protected from light→ rapid oxidation of bilirubin to biliverdin or
hydrolysis to free bilirubin.
-Ascorbic acid combines w/ diazonium salt→ prevents rxn w/ bilirubin.
 OTHER QUALITATIVE TEST FOR BILIRUBIN
Test Reagent Method/ Principle Interpretation
DIAZO TABLET • Confirmatory test for bilirubin
TEST (ICTOTEST • Principle: azocoupling reaction w/ a diazonium salt on which the reagent
METHOD) strips are based (Thus, same interferences w/ reagent strip)
• Sensitivity: 0.05-0.1 mg/dL
• Method:
1. 10 drops of urine are added to a special absorbent pad. Results:
2. Place ictotest tablet on top of the pad + 2 drops of water.
• (+) result: purple/ blue on the absorbent pad.
3. After 30 seconds→ tablet is removed
• (-) result: Any other color such as red or pink (still
4. Observe
negative).
ROSENBACH’S Nitric acid • Principle: Presence of an acid= bilirubin undergoes varying degrees of Results:
MODIFICATION oxidation. • (+) result: play of colors (green towards the urine
OF GMELIN’S • Method: layer)
TEST 1. Pipette 5ml of freshly voided urine in 13x100 mm test tube. • (-) result: play of colors without the green
2. Tilt the tube a little and overlay with 2 ml of nitric acid by allowing the
acid to flow on the sides of the tube.
3. Carefully return the test tube its original vertical position.
HARRISON’S • 10% Barium Chloride • Principle: Barium chloride combines with sulfate radicals in urine→ form
SPOT TEST • Fouchet’s Reagent barium sulfate precipitates (to which bilirubin will adhere).
(25% Trichloracetic acid, • Ferric chloride, in the presence of trichloroacetic acid, oxidizes bilirubin to
10% Ferric Chloride) biliverdin resulting to a color change.
• Method:
1. Pipette 5 ml of urine into 16x150 test tube.
2. Add 5 ml of 10% Barium Chloride.
3. Invert tube 3x to mix preparation.
Results:
4. Filter the preparation. (SAVE filtrate for urobilin determination)
5. Spread the filter paper and allow to partially dry. • (+) result: blue to green color
6. Place the filter paper into an evaporating dish then add a few drops • (-) result: no blue to green color
of Fouchet’s reagent at the center of the filter paper.