2ND SEMESTER – CLASS CODE

A.Y. 2022 – 2023 | MLS 321 ANALYSIS OF URINE AND BODY FLUIDS
UNIT 2: GLUCOSE, OTHER REDUCING SUGARS AND KETONES

Laboratory
Summary
I. Tests for Glucose in Urine
A. Reagent Strip tests
B. Copper Reduction Tests (Benedict’s Test)
C. Other Qualitative Tests
1) Fehling’s Test
Determination
2) Haine’s Test
3) Trommer’s Test sugars are not normally excreted in urine
4) Nylander’s Test • only glucose and galactose signify pathologic conditions.
5) Moore – Heller’s Test • Glucose is the most commonly encountered sugar.
6) Phenylhydrazine Test (Osazone Test) • In children below (2) years, test for galactose since it is
7) Kowarsky Phenylhydrazine Test commonly implicated in galactosemia.
(Blumel’s modification)
• Recommended: Fasting prior to the collection of samples
8) Glucose Oxidase Paper Strip Test
for glucose screening tests.
D. Other Quantitative Test
1) Benedict’s Test • Note:
2) Fehling’s Test A first morning specimen does not always represent a
3) Robert Fermentation Test fasting specimen because glucose from an evening meal
4) Somogyi method may remain in the bladder overnight, and patients should
be advised to empty the bladder and collect the second
II. Test for Other Reducing Sugars
specimen.
A. Fructose
• Conditions:
1) Seliwannof’s Test
2) Borchardt’s Test Diabetes monitoring Diabetes mellitus /
3) Barfoed’s Test Hypoglycemia
B. Lactose (Normal urine excretion: 20 mg/24 hrs) • specimens are usually Urine glucose testing in
1) Rubner’s Test tested two hours after conjuction with blood
2) Ormsby’s Test meals samples during the
3) Mucic acid test • 2-hour postprandial course of glucose
4) Phenylhydrazine / Osazone Test specimen tolerance tests
5) Woehl’s Test
C. Pentose (xylose, arabinos and ribose) • Detection of ketone
1) Bial – Orcinol Test - valuable monitoring and management tool for patients
2) Tauber’s Test with type 1 diabetes mellitus.
3) Tollen’s Test
4) Cole’s Test Ketonuria
5) Woehlk’s Test
- early indicator of insulin deficiency
6) Aniline Test
7) Phenyhydrazine / Osazone Test
Ketoacidosis
III. Test for Ketone - can develop slowly and progressively as a result of
A. Reagent Strip test repeated insufficient insulin doses.
B. Nitroprusside Tablet Test for Ketones
(Acetest)
C. Other Specific Tests for Ketones
1) Acetone
a) Rothera’s Test (also for Diabetic
acid)
b) Lieben’s Iodoform test
c) Gunning’s Test (modification of
Lieben Test)
d) Frommer’s Test
e) Lange Test
f) Jackson-Taylor's Test
(Modification of Legal's Test)
g) Diffusion test for Acetone
h) Wallhausser's Test
2) Diabetic Acid
a) Gerhardt's Test
b) Lindemann's Test
c) Arnold's Test
3) Beta – Hydroxybutyric Acid
a) Hart's Test
b) Osterberg test

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SUBJECT XX SEMSTER | [TRANS] / [BOOK] TITLE

TESTS FOR GLUCOSE IN URINE COPPER REDUCTION TESTS

(BENEDICT’S TEST – QUALITATIVE)
REAGENT STRIP TESTS • depends on the ability of reducing substances to
• uses a double sequential enzyme reaction convert cupric sulfate to cuprous oxide in the presence
of alkaline environment and heat
• detects glucose only

• Reducing substances:
- Glucose
- Fructose
- Galactose
- Lactose
- Maltose
• Glucose oxidase impregnated on the reaction pad rapidly - Pentose
catalyzes the oxidation of glucose to form hydrogen - Ribose
- Arabinose
peroxide and gluconic acid.
• The hydrogen peroxide formed oxidizes the chromogen • Color change result: blue à green à orange
(potassium iodide) on the pad into iodide in the presence of • Benedict’s reagent composition:
peroxidase - copper sulfate
• Color change: blue to green to brown - sodium carbonate
- sodium citrate buffer

• Benedict’s reagent composition:

Copper sulfate 17.3 g
• Reporting of results should include: Sodium citrate 173 g
o Plus system (negative / positive) Sodium carbonate 100g
- 1+, 2+, 3+, 4+
(anhydrous)
o Quantitative concentrations in mg/dL or g/dL
Distilled water --- making it 1L

• Brand variation
• Result
Brand Sensitive
Result Interpretation
Chemstrip 40 mg/dL
(-) No change from the original
Multistix 75 – 125 md/dL
blue color
vChem 45 mg/dL
Trace Green
Note: - Without any
sensitivity of the reagent strip is affected by urine precipitate
temperature, pH and specific gravity.
+ Greenish Yellow
- With yellow
What decreases sensitivity of strip?
precipitate
o ⇧ specific gravity
++ Yellowish green
o ⇧ pH
- With yellow
o ⇩ Temperature
precipitate
o Ketonuria (with ketone concentration ≥40
+++ Yellowish orange
mg/dL)
- Orange precipitate
++++ Orange red
• Interferences:
- With brick red
Ascorbic acid precipitate
- oxidized by the hydrogen peroxide and will, therefore,
compete with the oxidation of potassium iodide,
resulting in the inhibition of the color formation.

False Negative False positive

occur in the presence of occur in the presence of
ascorbic acid at a strong oxidizing agents (e.g.,
concentration of ≥50 mg/dL. sodium hypochlorite and
peroxides)

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SUBJECT XX SEMSTER | [TRANS] / [BOOK] TITLE

CLINITEST REAGENT TABLET o Reoxidation can occur:

• tablet version of Benedict’s test 1) presence of high concentrations of
• detect a minimum glucose concentration of 200 reducing substances
mg/dl. 2) following exposure of the reaction
• CLINITEST is a self- heating method for the mixture to room air after the protective
semiquantitative determination of reducing substances in CO2 gas blanket disperses.
the urine. • occur so rapidly that it can be missed if not observed
• Reagents: closely.
• If this occur (bright orange àdark brown), report the result
Reagent Function
as being greater than 2%. If measurement beyond 2% is
Citric acid Effervescent
medically desirable, an alternate two-drop method is
Sodium carbonate
available.
Sodium hydroxide • provides the alkaline
medium necessary for Two – drop method
the reaction • adding only 2 drops of urine to 10 drops of water, but a
Sodium hydroxide with • Provides alkaline special color chart is used.
water and citric acid medium • allow for quantitation up to 5% but the “pass-through”
Copper sulfate • reducing substances in phenomenon may still occur when very large
the urine then react with concentrations of sugar are present.
the copper sulfate to
reduce the cupric ions
to cuprous oxide.

• when placed in a mixture of water and urine, the tablet is

rapidly dissolved
• Heat is provided by the reaction of sodium hydroxide with
water and citric acid.
• CLINTEST procedure:
1) Place five drops of urine into a glass test tube (or use
0.3 mL)
2) Add 10 drops of water (or 0.6 mL) and mix by shaking
3) Drop one CLINITEST tablet into the tube and observe
the complete reaction.
4) Do not shake the tube during the reaction or for 15
seconds after the boiling has stopped.
5) At the end of the 15-second waiting period, shake the
tube gently and then compare
with the color chart that is provided.
6) The test is reported as negative, 1/4 % (or trace),
1/2% (1), 3/4% (2), 1% (3), or 2% (4).
• Determine whether positive copper reduction test is due
NEG Trace + ++ +++ ++++ to the presence of glucose or another reducing substance
0% ¼% ½% ¾% 1% 2% - both the glucose oxidase test and the reduction test
must be performed and a correlation made of the
results.
Blue Dark Light Green- Orange – Orange
green Green Yellow Yellow to red CLINTEST & REAGENT STRIP
Result Present component
• CLINITEST results must be evaluated immediately (+) Clintest Glucose
- any color change that occurs after 15 seconds is (+) Reagent strip
ignored. (-) Clintest Small amounts of glucose
- After bubbling ceases, the color observed (after the (1+) Reagent Strip
pass-through) may no longer indicate the high glucose (+) Clintest Non – glucose reducing
concentration, and falsely low results may be (-) Reagent strip sugar
reported. (-) Clintest False positive
(4+) Reagent Strip
Pass – through phenomenon
• occurs in the presence of high concentrations of reducing
substances False negative False positive
• evidenced by the color of the mixture “passing through” None if procedure is done Nalidixic acid
all colors possible properly
• Occurs when >2 g/dL sugar is present cephalosporins
• it becomes orange (the highest concentration) but then Probenecid
proceeds to change back to green-brown (a low ascorbic acid
concentration). urinary preservatives
• Mechanism: - formalin
- reoxidation of the resultant cuprous oxide to - formaldehyde.
cupric oxide and other cupric complexes (green).

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2ND SEMESTER – CLASS CODE
A.Y. 2022 – 2023 | MLS 321 ANALYSIS OF URINE AND BODY FLUIDS
UNIT 2: GLUCOSE, OTHER REDUCING SUGARS AND KETONES
GLUCOSE
Tests Method Procedure / Reagent Results and interpretations
Qualitative Tests
⭐⭐⭐⭐Fehling’s Test Reagent • Depends on the ability of glucose and other reducing sugars to
Fehling’s A Fehling’s B reduce alkaline copper sulfate reagent
Cupric sulfate in distilled water - Rochelle Salt - cupric hydroxide à cuprous hydroxide.
o Sodium potassium
tartrate + Yellow precipitate
o sodium hydroxide - Absence of yellow precipitate
o distilled water

1) Pipette 1mL each of Fehling’s A and B into 16x150 tubes

2) Add 4 ml of distilled water and boil the mixture for few seconds. N.B. If the mixture remains
clear after boiling, the solution may be used; otherwise, discard.
3) Continue boiling the solution at the same time, add 1 ml of the urine to be tested drop by
drop at the rate of 1 drop every 2 seconds

Haine’s Test Reagent: • Depends on the ability of glucose and other reducing sugars to
- Copper sulfate reduce alkaline copper sulfate reagent
- NaOH - cupric hydroxide à cuprous hydroxide.
- Glycerol
- distilled water
• Positive result: Yellow to red precipitate
Procedure:
- Four (4) mL of Haine’s solution is boiled and 6 – 8 drops of urine is added while keeping the
reagent but not boiling.
Trommer Test Reagent: • Depends on the ability of glucose and other reducing sugars to
- 10% KOH reduce alkaline copper sulfate reagent
- 10% Copper sulfate - cupric hydroxide à cuprous hydroxide.

Positive Negative
Yellow red precipitate Absence of yellow precipitate

• Positive: Yellow cuprous hydroxide or red precipitate

- It may spread out the entire field and a red sediment is then
formed
***If this reaction takes place upon heating, repeat the test
without heating.
Nylander’s Test Nylander’s reagent: • Based on the reduction of bismuth to metallic bismuth in a hot
- Rochelle Salt (Na or K lactate) alkaline solution.

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SUBJECT XX SEMSTER | [TRANS] / [BOOK] TITLE

- 10% NaOH or KOH

- bismuth subnitrate Negative White
(presence of phosphate)
Positive (+) Black color
1. Pipette 5 ml of urine into 16x150 test tube. Trace Brown color
2. Add 0.5 ml of Nylander's reagent. Mix the preparation. • ** Albumin in large amount may give similar reaction but may be
3. Allow the mixture to stand for 3-5 minutes. removed by boil and filter the urine with Nylander's reagent.
• If a Black solution is retained after boiling and filtration, the
substance present is sugar NOT albumin.

⭐⭐⭐⭐Moore Heller’s Method Reagent: • based on the caramelization of sugars by strong alkali and
- 10% KOH heat.

1. Pipette 2 ml of urine into 13x100 test tube. ≤1% Canary Yellow

2. Add 1 ml of 10% KOH. 1 – 2% Wine yellow
3. Boil the upper part of the mixture for 2-3 minutes. If phosphates are present in large 2 – 3% Cherry yellow
amount, filter the preparation and examine the filtrate. 3 – 4% Rum color
>4% Dark brown or black color

Phenylhydrazine Test (Osazone Reagent: • based on the principle that each sugar forms a definite form
Test) - Phenylhydrazine chloride when it comes in contact with phenyl hydrazine and acetate
- Sodium acetate (crystals separate when sugar is present).

• Positive result: phenylglucosazone crystals appearing as

Kowarsky Phenylhydrazine Test
(Blumel’s modification)
Glucose Oxidase Paper Strip Test
fine bright yellow needles arranged in bundles, sheaves
or rosettes.
Reagent: • Positive result: phenylglucosazone crystals appearing as
- Pure phenylhydrazine
fine bright yellow needles arranged in bundles, sheaves
- glacial acetic acid
- saturated solution of NaCl or rosettes.
Reagent:
- O – toluidine (indicate reagent) Positive Negative
Purple color in test area Remain Red
**can be false negative in
presence of ascorbic acid

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SUBJECT XX SEMSTER | [TRANS] / [BOOK] TITLE

Tests Method Procedure / Reagent Results and interpretations

OTHER QUANTITATIVE TESTS • glucose and other reducing substances reduce the copper sulfate to cuprous oxide and in the • Endpoint is reached when all blue color is gone and only a
presence of potassium thiocyanate is changed to white copper thiocyanate. gray color remnant
Benedict’s test • Reagent
Copper sulfate (pure Main reagent
crystallized)
Sodium carbonate provides alkaline medium
Trisodium citrate • Used to prevent the precipitation of cupric ions as cuprous
carbonate or cuprous hydroxide by forming a loosely bound
0.01 gram of glucose reduces 5 ml of Benedict’s reagent
complex with copper
( trisodium citrate: cupric complex ) which on Normal value 10.01 – 0.03g/100mL
dissociation gives a continuous supply of copper ions. 0.05 – 1.5 g/24 hrs
Potassium sulfocyanate • Precipitates cuprous ions as cuprous cyanide, a white colored
precipitate
Potassium ferrocyanide • prevent precipitation of cuprous oxide
Distilled water

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 disappear and a gray color remains

• N.B. If less than 1 ml of urine is used up to the end point, make a 1:5 dilution with distilled water
and repeat the procedure.
- In the computation, multiply the number of mI used up to the end point by the dilution factor.

4. Record the amount of urine used up to the end point and compute using the following formula.

Fehling’s test 1 ml of Fehling’s solution is mixed with 4 ml of distilled water and then heated to boiling. Urine is
added drop by drop until the grayish white color is obtained.

Robert Fermentation test

Somogyi method

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SUBJECT XX SEMSTER | [TRANS] / [BOOK] TITLE

Tests for Other Reducing Sugars

A. Fructose Procedure / Reagents Results
Seliwannof’s test⭐⭐⭐⭐
• Hot HCl converts fructose à hydroxymethyl furfural
+ Intense orange - red color
- links with resorcinol to form a red colored compound
- Dark precipitates (dissolves with
• Note:
ethanol)
- Concentrated HCl must not be more than 12 %
- Absence of red orange color and dark
- Reaction must be observed not after more than 20-30 seconds of boiling precipitate
- Glucose must not be present in amount exceeding 2 % because it can be converted
to fructose by action of HCl which may give false positive result.

• Reagent:
- Resorcinol
- Concentrated HCl
Borchardt’s Test
• Hot HCl convert fructose à hydroxymethyl furfural
Positive:
- links with resorcinol to form a red colored compound • Red precipitate after heating the urine sample with HCl and
• Reagent: resorcinol
- potassium hydroxide • Yellow – colored acetic ether layer after reaction with KOH and
- acetic ether acetic ether
Barfoed’s Test • Fructose reduce: Cupric acetate à cupric hydroxide à cupric oxide Positive:
• Confirmatory test: • Red precipitate
- addition of 10 ml water and 1 ml of phosphomolybdic acid is advised
Confirmatory positive: (after phosphomolybdic acid addition)
- very dark, almost opaque, blackish hue/color
Reagents
- cupric acetate
- 38% acetic acid
- distilled water
B. Lactose Procedure / Reagents Results
Rubner’s Test⭐⭐⭐⭐ Reagents: • Brick red solution with cherry red or copper colored precipitate
- Lead acetate
- concentrated NH4OH • Glucose will give yellow solution with yellow precipitate

1. Pipette 3mL urine into 13 x 100 test tube

2. Add 2mL of ammonia
3. Add 8 drops of 10% KOH
4. Warm in water bath (DO NOT BOIL)
Ormsby’s Test Reagents: Intense Red color
- Methylamine hydrochloride
- Na OH
Mucic acid test Reagents: Fine white precipitate of mucic acid crystals (also indicated increased
- Concentrated HNO3 galactose in urine)

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Phenylhydrazine / Osazone Test Reagents: • Formation of lactosazone crystals appearing as cotton-

- Phenylhydrazine hydrochloride ball-shaped (hedgehog)
- sodium acetate • Galactose in urine yields the same positive

Woehlk’s Test Reagents: Red color

- NH3
- 15% KOHN
C. Pentose Procedure / Reagents Results
Bial – Orcinol Test Reagents: Olive green solution in amyl alcohol
- Orcinol
• By Heating with HCl, pentose - concentrated HCl
is converted to - 10% FeCl3
furfural which reacts with
orcinol to form a colored
compound
Tauber’s Reagent
- Benzidine + Pink to red (cherry red)
Tauber’s Test⭐⭐⭐⭐ - glacial acetic acid - Absence of pink to red color
Other sugars Brown color after 9 – 10
minutes
***disregard any yellowish to brown color

Tollen’s Test Reagents: Cherry red color

- HCl
- phloroglucinol
Cole’s Test Reagents: Green Color
- Blood Charcoal
• Charcoal removes all reducing
substances except pentose and
formaldehyde
Woehlk’s Test Reagents: Red color
- NH3
- 15% KOHN
Aniline Test Reagents: • Bright red color in chloroform layer
• Sensitivity is 0.03% - Glacial acetic acid • Glucose fives an interfering green color
- pure aniline
- chloroform
Phenylhydrazine / Osazone Test Reagents: • Pentosazone crystals which appears as mass of entangles
- Phenylhydrazine HCl rootlets
- sodium acetate

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SUBJECT XX SEMSTER | [TRANS] / [BOOK] TITLE

KETONES
Test Procedure Results
A. Reagent Strip • based on the reaction of ketones with sodium nitroprusside (nitroferricyanide) impregnated in the
reagent pad.
• alkaline medium, acetoacetate reacts with nitroprusside to produce a color change from
beige to purple.
• Incorporation of glycine in the reagent pad enables detection of acetone.

False positive False negative

Sulfhydryl (2- mercaptoethane sulfonic rapid volatilization of acetone at room
acid, captopril, cysteine) temperature
levodopa metabolites breakdown of acetoacetate by bacteria
- urine specimens should be tested
immediately or refrigerated
phenylketones deterioration of the nitroprusside
- cause red-orange coloration reagent in the reaction pads from
Phthaleins exposure to:
- bromsulphthalein - moisture
- phenolsulfonphthalein red dyes - heat
High specific gravity - light
low pH
ACETEST (nitroprusside Tablet • Detect:
Test) 1. Place the tablet on a piece of clean, dry white paper. - 5 -10 mg/dL of diacetic acid
• 10 times more sensitive to 2. Put one drop of urine directly on top of the tablet. - 20 - 25 mg/dL of acetone
diacetic acid than to acetone 3. Compare the color of the tablet with the color chart at 30 seconds. • Not react with beta hydroxybutyrate
4. Report:
Small 5 – 10 mg/dL of diacetic acid
Moderate 30 – 40 mg/dL
Large 80 – 100 mg/dL
• Reagent
- sodium nitroprusside
- glycine
- a strong alkaline buffer (disodium phosphate)
- Lactose Positive Negative
o helps enhance the color. Purple Pink
• Diacetic acid and acetone react with sodium nitroprusside and glycine in an alkaline medium to Tan
form a purple color. Yellow
OTHER SPECIFIC TESTS FOR KETONES
A. ACETONE Procedures Results
Rothera’s Test • based on the decomposition of sodium nitroprusside or sodium ferrocyanide to sodium
(Also for Diacetic acid) ferrocyanide and ferric oxide in an alkaline solution.
• nitroprusside reaction is 20 times more sensitive to acetoacetate than acetone Positive Negative

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SUBJECT XX SEMSTER | [TRANS] / [BOOK] TITLE

• does not react with beta-hydroxybutyrate. Red to purple ring at zone Purple ring
• False positive: of contact
- after a heavy meal (color persists for 30 seconds then disappear or fade within 3-4 minutes)
o In the same way, amorphous urates give a brown or orange color.

Lieben’s iodoform test • Reagent: Yellow precipitate of iodoform

- KOH - thin, yellow hexagonal plates or starlike groups of
- Iodine crystals.
- potassium-iodine solution
Gunning’s Test (Modification of • same with the Lieben’s test • Black color indicates formation of nitrogen iodide but
Lieben Test) - except that it involves addition of an excess alcoholic iodine solution and ammonia disappears
water. • Iodoform precipitate is formed when acetone is present
Frommer’s Test • reaction of one molecule of salicylic aldehyde with one molecule of acetone to form purple/purplish red ring at the junction of the two liquids
oxybenzol acetone
- in the presence of strong alkali forms dioxy-dibenzol acetone forming a red-purple color-
usually intense purplish red ring

• Reagents:
- KOH
- 10 % alcoholic solution of salicylaldehyde

• Procedure
1) Glacial acetic acid and saturated solution of sodium nitroprusside is added with 5 ml of
urine.
2) 28 % ammonium hydroxide is overlayed
3) a purple/purplish red ring at the junction of the two liquids is considered as a positive result.
Legal’s test⭐⭐⭐⭐ • based on the decomposition of sodium nitroprusside or sodium ferrocyanide to sodium Purple or violet red color
ferrocyanide and ferric oxide in an alkaline solution.

1. Place a few ml of freshly voided urine sample in 16x 150 test tube.
2. Add enough NAOH or KOH to render the sample alkaline. Test with litmus paper after each
addition of alkali. • Alcohol and acetic aldehyde and diacetic acid gives the same
3. Add a few drops of fresh Sodium Nitroprusside solution. reaction
4. Add a few drops of concentrated acetic acid.

Lange Test • Glacial acetic acid and saturated solution of sodium nitroprusside is added with 5 ml of urine. Purple/purplish red at the junction of the two liquids
• 28 % ammonium hydroxide is overlayed
• a purple/purplish red ring at the junction of the two liquids is considered as a positive result

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Jackson – Taylor’s Test 1) Few drops of sodium nitroprusside are added in a few ml of urine. few purple-red color or permanganate colored ring at the point of
(Modification of Legal’s Test) 2) Ammonium sulfate is added followed by ammonium hydroxide. contact indicates the presence of acetone
Diffusion test for acetone • One (1) ml of Nessler’s reagent in a small petri dish is acidified with glacial acetic acid or HCl. cream colored precipitate

• Acetone in the urine being volatile diffuses and is fixed inside the covered petri dish which
could react 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 cyanide, silver nitrate, and NaOH)
• Scott – Wilson Reagent
- Mercuric cyanide
- Silver nitrate
- NaOH
B. DIACETIC ACID
Gerhardt’s Test • Ferric ions chelate with enol groups of diacetic acid Bordeaux red or wine-red complex
• FALSE POSITIVE - indicates presence of diacetic acid and or interfering
- aspirin (salicylates) substances like drugs
- phenol
- antipyrine
- sodium bicarbonate.
Lindemann’s Test Reagents:
- 30 % acetic acid Positive Negative
- iodine No change of color Reddish violet
- chloroform
o will become reddish violet if diacetic is not present. • Chloroform will become reddish violet if diacetic is not present
Arnold’s Test Reagent: Violet color
- (1) % sodium nitrate
- acetophenone solution
- ammonia
- concentrated HCl
C. Beta – hydroxybutyric acid
Hart’s Test
• Boiling breaks down diacetic acid to acetone and acetone is removed by evaporation. POSITIVE NEGATIVE
Addition of hydrogen peroxide oxidizes remaining acetone and diacetic acid. Tube with Tube without
hydrogen peroxide hydrogen peroxide
red ring at the point of No change in reaction
1) Place 5 ml of freshly voided urine sample in 16x150 test tube.
contact indicates presence
2) Add equal volume of water and a few drops of acetic acid.
of beta- hydroxybutyric
3) Boil the preparation ½ its original volume.
4) Dilute to 10 ml with distilled water. acid
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.

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Osterberg Test Reagent:

- NH4OH >0.5% <0.5%
- sodium nitroprusside Purple color is deeper in Color is lighter
- distilled water shade than the standard

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