Integrated Science - Published in CHEM 13 NEWS

When I was a teacher at John McGregor Secondary School in Chatham, Ontario, I had some articles published in CHEM 13 NEWS of the University of Waterloo . Here is one that appeared in two parts in the December 1980, and in January 1981 issues:

Integrated Science
A unit for use by High School Science Students

Thomas A. Vella-Zarb
John McGregor Secondary School, 300 Cecile Avenue, Chatham ON N7M 2C6

In this unit you will be using:
a) skills acquired in physics, such as density of liquids,
b) concepts learned in chemistry, such as precipitation of insoluble substances,
c) principles of health, such as the function of the kidneys,
d) topics learned in biology, such as excretion.

This unit would be of great benefit to those students who intend to pursue careers in health, such as medicine, nursing, pharmacy, public health, and laboratory technology. The unit is very practical, and because it deals with common everyday human problems, it should also be interesting to other students. Most of the equipment needed is readily available in the chemistry laboratory of any high school. If the tests are carried out at home, some improvisation may be necessary, or the equipment may be signed out from the school laboratory. The equipment used includes:

Bunsen burner, hydrometers, pipettes, eyedroppers, mortars and pestles, beakers, graduated cylinders, flasks, crucibles, test tubes, evaporating dishes, pH test paper, and similar items.


Using reference books, such as biology texts, health books and encyclopedias, find out about the function of the human kidney. From your readings answer the following questions:
1. What relationship is there between the volume of ingested fluids and the volume of excreted urine?
2. What relationship exists between the volume of fluids circulating in the human body and the excreted fluids?

Examine a sample of fresh urine. Make a note of its appearance, odour, and colour. Compare your findings with the normal properties:

Appearance: At the time of voiding, it should be clear.
Odour: A faint aromatic odour is usually present.
Colour: Usually a deep yellow colour is observed, due to the presence of urochrome.

Variations in appearance may indicate cause for concern, or on the other hand may not mean anything much. For example, after a rich meal, the urine may be turbid, due to the urine turning alkaline. Fats present in the urine may also tend to make the urine turbid or cloudy. If a clear sample of urine is left to stand for a while, cloudy opacities (nubeculae) formed by the action of mucin from the urinary passages and phosphates, etc., may appear.

After ingestion of asparagus, the odour of urine changes completely. Consumption of some substances - drugs, foodstuffs, etc. - may cause a change in the colour of urine; thus, red urine may (besides the obvious presence of blood), indicate the ingestion of aniline dyes in some candies, or the taking of drugs such as Pyridium (Phenazopyridine Hydrochloride), Azo-Gantrisin or aminopyrine. After ingesting Azuresin (Diagnex Blue) as a test for stomach cancer, abnormal gastric acid secretion, and gastric polyps, the urine may turn blue. A few instances of pale blue fluorescence in urine have been reported after patients ingested Triamterene (Dyrenium) to relieve oedema in congestive heart failure, and the like. When the urine is dark brown, this usually indicates alkaptonuria, hemoglobinuria, poisoning by phenol or cresol, or melanin in the urine.


Since the students carrying out these tests are adults, the values for the normal human adult are given.
The volume excreted in twenty-four hours is 600 to 1600 cm3, and its specific gravity; 1.001 to 1.030.

The pH of urine ranges from 5.53 to 6.97, with 6.25 being a mean.

A test devised by Volhard for volumes of urine has the subject drinking 1000 cm3 of water after emptying his/her bladder. Elimination is then carried out every hour and a record is kept. Normal values obtained by Volhard show the following:

1 140 1.008
2 500 1.002
3 270 1.003
4 90 1.010
Total volume in the first four hours is 1000 cm3 6 40 1.020
8 45 1.026
10 85 1.025
12 30 1.031


In this test the subject takes the specific gravity and the volume of urine excreted according to the following table, and interprets the results according to the notes below.

Time Volume (cm3) Specific gravity
10.00 am
12.00 noon
2.00 pm
4.00 pm
6.00 pm
8.00 pm
8.00 pm to 8.00 am

Usual values for the night sample are between 250 and 500 cm3. This value should never exceed 750 cm3 in a normal person. Its specific gravity should be about 1.018. During the day, one of the specimens should have a specific gravity over 1.018. The difference between the highest and lowest specific gravity findings should not be less than 8 to 9 points. (Note: Points in this case refer to the last two digits after the decimal.)

If there is impairment of renal function, the night sample volume 750 cm3 or more would indicate nocturnal polyuria. Another indication of impaired renal function is evident when the difference in the specific' gravity of the various samples is less than 8 or 9 points. In this case, the kidneys are losing their power to concentrate the urine. (The figures have to be adjusted if there is significant albumin or sugar present in the urine.)


The subject takes a normal supper in the evening before the test, making sure that it contains a large amount of protein but not more than 200 cm3 of fluid. No more food or drink is then allowed until the end of the test the following morning. Before retiring, the subject empties his/her bladder and this specimen is discarded.

The urine is then collected in three specimens as follows:

1. All the urine voided from the time of retiring to the time of awakening in the morning.
2. The subject stays in bed and a-.sample is collected one hour after the first specimen.
3. The subject stays in bed one hour more, and then a third specimen is collected.
Volumes and specific gravities for each specimen are recorded. Normally one specimen should have a specific gravity of at least 1.024.


A sample of urine is allowed to stand in a cylinder or a suitable sedimentation glass, and the sediment is then pipetted onto a slide for examination. If the urine appears to be quite clear, a sediment may be obtained more readily by centrifuging the sample. What one observes in the sediment may be grouped as follows.
l. Organizing elements such as erythrocytes, leucocytes, epithelial cells, and bacteria.
2. Casts from the uriniferous tubules.
3. Crystalline and amorphous chemicals such as uric acid, urates, oxalates, cystine, etc. (see the identification chart).
4. Miscellaneous items such as mucus, spermatozoa, parasites, and even foreign bodies, such as starch granules, hairs, and fibres.

Macroscopic examination plays an important part in the checking of urine samples. Chemical concepts about the behaviour of certain chemicals help identify some of the chemicals present in urine. For example uric acid and urates dissolve when warmed to 60C in alkaline solution. Phosphates dissolve in the presence of acetic acid, and so on.

From Harrison, G. A. "CHEMICAL METHODS IN CLINICAL MEDICINE", Fourth Edition, J. and A. Churchill, London, 1957, pp 100 et seq.


We may assume that the samples of urine provided by the students will be normal since it is presumed that the students are healthy. To make the tests more interesting and varied, it may be advisable to "doctor" the specimens in different ways tb get positive reactions. A drop or two of acetone can easily be detected in the acetone test. Proteins are plentiful. Squeezing a little juice from some raw meat into the specimen or adding a tiny bit of egg white will give a guud positive result for the protein-in-urine test. When doctoring for sugars you must remember that the sugar test is for reducing sugars, and so dextrose, fructose, and lactose are to be added - not sucrose.


Protein or albumin is not normally present in urine. When albumin is found, the condition is known as albuminuria, and this indicates a flaw in the filtering system of the kidneys. Various tests are . available to detect albumin in the urine.


Pour a sample of urine into a pyrex test tube (about 10 cm3 is sufficient). Heat the upper portion of the sample over a flame.
The formation of a cloudy precipitate indicates either phosphate salts or albumin.
To differentiate between the two, add a few drops of acetic acid, 5%.
If a precipitate persists, this shows that albumin was present. If the precipitate dissolves, then phosphate salts were present.


Mix equal parts of urine and Exton reagent in a test tube. Absence of cloudiness indicates albumin. If the sample is cloudy this does not indicate a negative result. Check further. Heat the sample gently but do not boil. If cloudiness remains or increases albumin is present.


Pour about 5 cm3 of urine into a test tube. Add, drop by drop, a 20 to 25% solution of sulfosalicylic acid, and mix. If albumin is present, a white cloud will form. This turbidity is not cleared upon heating. This test is very sensitive, being able to detect protein in concentrations as low as 15 parts per million.


To 5 cm3 of filtered clear urine, add 0.5 cm3 Sorensen's acetate buffer solution and boil for about one-half a minute. A fine flocculation indicates the presence of more than traces of albumin. (Note: phosphates and urates do not give any flocculation with this test.)


When Albumatest tablets are dissolved according to directions on the commercial package, they form a solution of sulfosalicylic acid, and sodium bicarbonate. Equal portions, usually about 10 drops, of urine and tho prepared solution are mixed together and a positive reading is made according to the amount of cloudiness or prrcipitation that forms.


This test, when positive, is a significant diagnostic aid in multiple myeloma - a malignant tumour in the bone marrow. Pour 10 cm3 urine into a test tube. Place this test tube and a thermometer into a beaker of water. Heat gently, observing both the urine and the thermometer. A positive Bence Jones is indicated by the following three conditions, all of which have to be met:

a) at 50 to 60, a white cloud appears which turns into a white precipitate, and which usually clings to the sides of the test tube.
b) adding a few drops of 5% acetic acid and further heating to boiling point, the precipitate will either partially or completely disappear.
c) when the urine is filtered while hot, using a heated funnel and, heated test tube, the white cloud will reappear as soon as the liquid cools to room temperature.



To 5 cm3 of urine, add 0.5 cm3 of Benedict's reagent. Boil vigorously for about two minutes. Positive readings vary from yellow to brick red precipitation.

Heat in separate test tubes, over the same flame, 5 cm3 of urine, and 5 cm3 of a mixture of Fehling's reagents I and II_ Avoid boiling. Then pour the heated Fehling's on to the heated urine, slowly. A yellow-red precipitate, which sometimes turns to green upon further heating, is a positive reading for sugars.


The use of this indicator tape for testing the presence of sugar depends on enzyme action. Compare the colour against a chart provided on the package


This is a commercially available tablet for diagnosing glycosuria. Put 10 drops of water, and 5 drops of urine into a tAgt toga. Put in one tablet and observe the reaction. Heat is generated, the liquid boils, and reactions similar to Fehling's or Benedict's occur due to the copper(II) sulphate present in the tablets.


A woman's pre-partum and post-partum samples of urine may give false positive test for sugar (glucose). Possibly this is due t4 the presence, just before and just after the birth of the baby, of lactose, which reacts similarly to glucose. To distinguish between the two, stir the urine sample, and pour 10 cm3 into a test tube. Add 3 g of lead acetate and after -baking well, filter. Boil the filtrate. Add 2 cm3 of concentrated ammonium hydroxide solution and boil again. If lactose is present, the solution turns brick red and a red precipitate forms. If glucose is present, the solution also turns red, but the precipitate is yellow.
For further enrichment, the student should find out about diabetes mellitus.


The presence of acetone is very significant, because it often indicates acidosis. When acidosis is present in the body, the alkali reserve is decreased, and this may be fatal. Untreated diabetes mellitus, some fever, diarrhoea, excessive vomiting, and other conditions where the metabolism of glucose is impaired may be detected by the presence of acetone in the urine


These are commercially available diagnostic tablets containing nitroprusside. A colour chart is provided on the side of the package, against which a reading is taken. One tablet is placed on a clean surface, such as a sheet of white paper. One drop of urine is then put on the tablet, and a reading is taken after 30 seconds. The colour is compared to the chart. (This test may also be used for plasma, serum, or even whole blood.)


Pour about 5 cm3 fresh urine in test tube. Add about 2 g solid ammonium sulphate, and mix well to saturate the urine. Add 2 drops of sodium nitroprusside reagent. Mix. Tilt the test tube to about a 30 angle and, using a medicine dropper, slowly , overlay the sample with 28% ammonium hydroxide. If a purple to a red colour appears at the junction of the two liquids, the test is positive. This colour may take up to 15 minutes to show, but it does not fade away once present. Disregard any brown or orange colours.


Pour about 5 cm3 urine into a test tube. Add, drop by drop, loo w/v solution of ferric chloride until no more precipitate forms. In the presence of excessive quantities of diacetic acid (i.e., acetoacetic acid), a Bordeaux red colour will develop. If acetylsalicylic acid is present, a purple colour will develop, but the colour may be deceptive, and confusion may arise. To confirm the presence of diacetic acid, boil 5 cm3 of urine in a pyrex test tube for 3 minutes. (Alternatively, place it in boiling water for 15 minutes). Any diacetic acid present will thus be converted to acetone, which will volatilize. After the sample cools, repeat the test with ferric chloride. If diacetic acid was originally present, the test should now be negative. If acetylsalicylic acid or similar drugs were present, the test will still be positive.


Should there be more than a few erythrocytes in the urine sediment, there is cause for concern, since their presence indicates bleeding within the urinary tract. The condition known as haemoglobinuria is present when occult (hidden) blood is present in the urine. This blood is not readily visible as it may be dissolved or haemolyzed. Excessive red cell destruction may occur because of a reaction during blood transfusion, as a result of severe burns, and in some chemical poisonings. These cause haemoglobinuria.


In this test the haemoglobin in the blood reacts with hydrogen peroxide liberating oxygen, which then reacts with an organic reagent producing a coloured compound. Mix the urine and pour about 2 cm3 into a test tube. Add 3 cm3 of a saturated solution of benzidine in glacial acetic acid and mix. Add 1 cm3 of a 3% solution of hydrogen peroxide and mix. A positive reaction is indicated by a green to blue colour.


Place about 1 cm3 urine in a test tube. Add 1 cm3 orthotoluidine reagent. Add about 10 drops of 30 hydrogen peroxide and mix. A positive reaction shows green to blue colour.


This is a convenient method, wherein the chemicals orthotoluidine and hydrogen peroxide are present in cellulose strips. The test is carried out by dipping the strip in a well mixed sample of urine, or briefly passing it through the urine stream. Reading is done after 30 seconds and the amount of occult blood present is estimated by comparison with the colour charts supplied by the manufacturers.


This is similar to the previous test and is manufactured by the same company. The chemicals are contained in tablets. Place one tablet on a filter paper. Place a large drop of urine on the tablet (so that the urine just flows over the sides.) A blue colour is a positive test. Again, the amounts may be read by comparison with the colour chart on the side of the container.


Students working with volumes and specific gravity or urine samples should be encouraged to find out the dry weight of urine. A normal adult excretes 55 - 70 g of solids in a twenty-four hour period. Obviously, one method of determining the dry weight would be experimentally by actually evaporating to dryness samples of urine, and then calculating from the results they find. They may be interested in a simple formula devised by Hoppe-Seyler:

Multiply the 2nd and 3rd figure after the decimal point of the specific gravity of urine, by 2.6 in the case of adults, or 1.6 in the case of small children. The result is the amount in grams of the dry weight of urine. e.g. S.G. 1.020, the dry weight would be: 20 x 2.6 = 52 g/24 hours


Samples of "stones" may be obtained from hospitals, [or veterinarians] if you have the right connections. Students are usually able to furnish some samples from relatives who have had them removed, either by surgery, or by passing them naturally (and painfully).

The first step in checking a calculus is to examine it physically - macroscopically. Make a note of its size, shape, colour, surface appearance, and consistency. If its mass is to be noted, make sure that the stone has been allowed to dry at room temperature for at least twenty-four hours.

The following chart helps identify the type of some stones one may encounter:

Type of StoneColour Surface Consistency
urateyellow to dark red- brownslightly roughquite hard
phosphatewhite, gray,yellow-graysmooth sandychalky, brittle
cystinepale yellowwaxlikesoft
oxalatewhite, gray, red-brown to black quite rough or tuberculatedvery hard
carbonate dull-white to red-brownchalky usually quite hard
xanthine brown smooth, waxy soft

After all observations have been made on the intact stone, the stone is prepared for chemical examination. The whole stone or part thereof is finely pulverized in a mortar with a pestle. The first test to be performed is usually a flame test. This is carried out in either of two ways.


In this method, a nichrome or platinum innoculation loop is first dipped in distilled water and then to the finely powdered calculus. The powder covered loop is then placed in an open colourless flame and observed. In summary the observations and their interpretations are as follows:

A. complete or almost complete ignition of the powder indicates that the stone is mainly organic.

B. no charring at all indicates that the stone is mainly inorganic.

C. charring, leaving behind a tarry mass, indicates sulphonamides as the main constituent of the stone.

D. a small of burnt feathers indicates the presence of fibrin.


A small amount of the powder is transferred to a crucible or to a platinum boat. This container is held in the flame of a Bunsen burner and observations made as above.


Put some of the residual ash from the crucible method into a test tube and add either dilute hydrochloric acid, or add 2 cm3 distilled water and 2 drops of concentrated hydrochloric acid. Effervescence indicates the presence of oxalates. If no residue remains after igniting the powder, the oxalate test may be carried out later.


Fill the test tube from above test with a saturated solution of ammonium oxalate. A heavy white precipitate indicates calcium. (Note: calcium is always present when oxalates are present, but the reverse is not always true,)


To a small amount of unignited powder add a few drops of dilute HCl. Effervesence indicates the presence of carbonates.


To the test tube from the above test add either of the following:

a) 3 cm3 distilled water, 20 drops molybdate reagent and 10 drops amino-naphthosulphonic acid reagent.

b) 2 drops of a 5% ammonium molybdate solution in dilute H2SO4. In either case, the appearance of a deep blue colour is positive for phosphates.


To some unignited powder add 10 drops of 10% aqueous sodium hydroxide. The odour, if any, is noted. An odour of ammonia indicates the presence of ammonium magnesium phosphate.


To some of the powdered stone in an evaporating dish add a drop of two of concentrated nitric acid. Strong foaming is presumed to indicate uric acid. To confirm this, evaporate to dryness - very carefully - over a steam bath. Cool to room temperature - the residue should be either yellow or red - and add two drops of concentrated ammonium hydroxide. If the colour turns to purplish-red, and fades when the crucible is heated again, this is definite proof of the presence of uric acid.


Either of two tests may be performed:

a) Take a drop of the slurry from the carbonate test. It this had been discarded, take some powder, add dilute hydrochloric acid to it, and from this take one drop. Place this drop on a ceramic tile. Add one drop of 5% sodium cyanide in dilute sodium hydroxide solution. Mix, and wait five minutes. Add one drop of sodium nitroferrrcyanide solution. An appearance of a red colour, which may fade after awhile, indicates the presence of cystine.

b) Put some unignited powder in a test tube and add 1 cm3 of a 10% sodium hydroxide solution. Heat this mixture in a water bath. Then add a few crystals of lead acetate and heat a little longer. If the mixture turns black, cystine is present.


One drop of ''slurry'' from the carbonate test above is placed on a ceramic tile. Add one drop of a nitrophenylazobenzene alcoholic alkaline solution and five drops of sodium hydroxide solution. A blue precipitate indicates the presence of magnesium.


To some powdered stone add about five drops of chloroform. A blue colour shows indigo.


Dissolve some powdered stone in nitric acid. Evaporate to dryness un a water bath. Add three drops of ammonium hydroxide solution. If the residue turns orange, it is a positive test for xanthine. (Compare this with the uric acid test above.)

OXALATE TEST (another method)

Some unignited powder and a few drops of dilute hydrochloric acid are mixed together. Put in two drops of manganese oxide suspension in dilute hydrochloric acid, Tiny bubbles evolve within five minutes for a positive test.


Although most of the reagents are readily available in the laboratory, a number of them have to be freshly prepared according to special recipes.


Pipette 5 cm3 glacial acetic acid into a 100 cm3 volumetric flask, dilute to volume with distilled water and mix throughly.


This is readily obtained from stock bottles.


Weigh 10 g of barium chloride and transfer to a 100 cm3 volumetric flask, dissolve it and dilute to volume with distilled water.


Dissolve 173 g sodium citrate, and 100 g anhydrous sodium carbonate in about 600 cm3 distilled water. In a separate container dissolve 17.3 g crystalline cupric sulphate in about 100 cm3 distilled water. The copper sulphate solution is then added to the other solution with constant stirring. Filter if not clear. Make up to 1 litre with distilled water.


Weigh 4 g benzidine powder and transfer to a brown bottle. Add 50 cm3 of glacial acetic acid. Shake to dissolve as much as possible. This solution is stable for about one month.


Put 10 g of anhydrous calcium chloride into a 100 cm3 volumetric flask. Dilute and make up to volume with distilled water.


Pour 150 cm3 distilled water into a brown bottle. Add 10 g paradimethylaminobenzaldehyde. Swirl the mixture, and then carefully add 150 cm3 concentrated hydrochloric acid. Mix.


Weight 200 g of sodium sulphate decahydrate and transfer to a litre volumetric flask into which you have previously poured 800 cm3 distilled water. Dissolve by swirling the flask. Add 50 g sulfosalicylic acid and dilute to volume with distilled water. Mix.


Weigh 10 g ferric chloride and transfer to a 100 cm3 volumetric flask. Make up to volume with distilled water, mixing thoroughly.


Transfer 25 g trichloracetic acid to a flask containing 100 cm3 distilled water. Add 10 cm3 ferric chloride solution (see above). Mix.


Weigh 5 g Iodine crystals and 10 g potassium iodide. Transfer to a brown bottle. Add 100 cm3 distilled water, and mix.


Dissolve 4 g orthotoluidine in 100 cm3 glacial acetic acid. This solution keeps for about one month if kept in a brown bottle stored in a refrigerator.


Add 40 g sodium nitroprusside (sodium nitroferricyanide) to 100 cm3 distilled water in a brown bottle. Shake to dissolve as much as possible. Leave any undissolved salt in the bottle to ensure a saturated solution.


Weigh 20 g sulfosalicylic acid and transfer to a 100 cm3 volumetric flask. Dissolve and dilute to volume with distilled water.


Weigh 2.5 g ammonium oxalate and 2.5 g oxalic acid. Transfer to a brown bottle. Add 145 cm3 distilled water, and 5 cm3 glacial acetic acid. Mix well.


Place ethyl alcohol in a beaker and add zinc acetate with constant stirring until no more dissolves.


Dissolve 25 g Ammonium molybdate in 200 cm3 distilled water, in a one-litre flask. Add 300 cm3 5 M sulphuric acid. Mix, and dilute to volume, very carefully, using distilled water.


Dissolve 30 g sodium bisulphite in 200 cm3 distilled water. Dissolve 20 g sodium sulphite in 100 cm3 distilled water. Filter separately. Put 195 cm3 of the bisulphite solution in a flask. Add 0.5 g aminonaphtholsulphate and 5 cm3 of the sodium sulphite solution. Stopper and shake well. If the salt does not dissolve, continue adding one cm3 of the sulphite solution at a time and shake until it all dissolves. Store in a cool dark place. Stable for one month.


I - 3.5 g copper(II) sulphate dissolved in 50 cm3 distilled water.

II - 17.5 g potassium sodium tartarate, and 5 g sodium hydroxide dissolved in 50 cm3 distilled water.

To use, equal parts of I and II are freshly mixed together.


56.5 g glacial acetic acid and 118 g sodium acetate are dissolved in 1 litre of distilled water.

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