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.

MICROSCOPIC EXAMINATION OF URINE

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 60°C in alkaline solution. Phosphates dissolve in the presence of acetic acid, and so on.

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

DOCTORING THE SAMPLES OF URINE

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

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.

HEAT AND ACETIC ACID TEST

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.

EXTON TEST

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.

SULFOSALICYLIC ACID TEST

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.

SORENSEN TEST

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.)

ALBUMATEST

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.

BENCE JONES TEST

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.

SUGARS

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.

FEHLING'S REAGENT
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.

TES-TAPE

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

CLINITEST

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.

RUBNER TEST

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.

ACETONE

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

ACETEST TABLETS

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.)

ROTHERA'S TEST

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.

LANGE TEST

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.

BLOOD AND BLOOD PIGMENTS

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.

BENZIDINE TEST

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.

ORTHOTOLUIDINE TEST

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.

HEMASTIX

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.

HEMATEST

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.

DRY WEIGHT OF URINE

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

ANALYSING URINARY CALCULI

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:

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.

INNOCULATION LOOP METHOD

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.

CRUCIBLE METHOD

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.

OXALATE TEST

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.

CALCIUM TEST

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,)

CARBONATE TEST

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

PHOSPHATE TEST

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.

AMMONIUM MAGNESIUM PHOSPHATE

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.

URIC ACID TEST

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.

CYSTINE TEST

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.

MAGNESIUM TEST

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.

INDIGO TEST

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

XANTHINE TEST

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.

REAGENTS

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

ACETIC ACID, 5%

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

AMMONIUM HYDROXIDE 28%

This is readily obtained from stock bottles.

BARIUM CHLORIDE 10%

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

BENEDICT'S REAGENT

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.

BENZIDINE SOLUTION, SATURATED

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.

CALCIUM CHLORIDE 10%

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

EHRLICH'S REAGENT

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.

EXTON REAGENT

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.

FERRIC CHLORIDE 10%

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

FOUCHER'S REAGENT

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

LUGOL'S SOLUTION

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

ORTHOTOLUIDINE REAGENT

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.

SODIUM NITROPRUSSIDE

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.

SULFOSALICYLIC ACID 20%

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

SULKOWITCH REAGENT

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.

ZINC ACETATE, ALCOHOLIC SOLUTION

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

MOLYBDATE REAGENT

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.

AMINONAPHTHOL SULPHATE

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.

FEHLING'S SOLUTIONS

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.

SORENSEN'S ACETATE BUFFER SOLUTION

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