Examination of urine

Possible pathogens in urine

Bacteria Gram positive Staphylococcus saprophyticus
Haemolytic streptococci
Gram negative Escherichia coli
Proteus spps
Pseudomonas spps
Kliebsiella spps
Parasites Protozoan T. vaginalis
Helminths S. haemotabium
E. vermicularis
W. bancrofti
O. volvulus
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COLLECTION & TRANSPORT OF URINE

• Whenever possible, the first urine passed by the

patient at the beginning of the day should be sent for
examination.
• This specimen is the most concentrated and
therefore the most suitable for culture, microscopy,
and biochemical analysis.
• Midstream urine (MSU) for microbiological
examination is collected as follows:

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 Collection… cont’
1. Give the patient a sterile, dry, wide-necked, leak-proof
container and request a 10–20 ml specimen.
– Important: Explain to the patient the need to collect the urine
with as little contamination as possible, i.e. a ‘clean-catch’
specimen.
– Female patients: Wash the hands. Cleanse the area around the
urethral opening with clean water, dry the area with a sterile
gauze pad, and collect the urine with the labia held apart.
– Male patients: Wash the hands before collecting a specimen
(middle of the urine flow).
– Note: When a patient is in renal failure or a young child, it may
not be possible to obtain more than a few millilitres of urine

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 Collection… cont’

2. Label the container with the date, the name and

number of the patient, and the time of collection.
– As soon as possible, deliver the specimen with a request
form to the laboratory.
– When immediate delivery to the laboratory is not possible,
refrigerate the urine at 4–6°C.
– When a delay in delivery of more than 2 hours is
anticipated, add boric acid preservative to the urine
– Specimens containing boric acid need not be refrigerated.

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 Deterioration of urine
• The following changes occur when unpreserved urine is
left at room temperature:
– Any bacteria in the urine will multiply so that the bacterial
count will be unreliable. When the organisms are urease-
producing, the ammonia released will increase the pH of
the specimen which will result in the destruction of cells
and casts. Bacteria will also break down any glucose which
may be present.
– When white cells, red cells, and casts are present, these
will begin to lyze especially in a concentrated specimen.
– The concentration of protein in the urine will be altered.
When bilirubin is present this may be oxidized to biliverdin
which will not be detected. Likewise, urobilinogen will not
be detected because it will be oxidized to urobilin.

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 Laboratory examination of urine
• Day 1
1. Describe the appearance of the specimen
• Report
– Colour of the specimen
– Whether it is clear or cloudy (turbid)

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 Normal freshly passed urine is clear and pale yellow
to yellow depending on concentration
Appearance Possible cause
Cloudy Bacterial urinary infection
Red and cloudy Urinary schistosomiasis
Bacterial infection
Brown and cloudy Malaria heamoglobinuria
Other conditions for intravascular
hemolysis
Yellow brown or green Acute viral hepatitis
brown Obstructive jaundice
Yellow orange Hemolysis
Hepatocellular jaundice
Milky white Bancroftian filariasis
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 Day 1…. Cont’

2. Examine the specimens microscopically

• Urine is examined microscopically as a wet
preparation to detect:
– Significant pyuria, i.e. WBCs in excess of 10 cells/µl of
urine
– Red cells
– Casts
– Yeast cells
– T. vaginalis motile trophozoites
– S. haematobium eggs
– Bacteria (providing the urine is freshly collected)

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 Preparation and examination of a wet
mount
1. Aseptically transfer about 10 ml of well mixed urine to a labelled conical
tube.
2. Centrifuge at 500–1000 g for 5 minutes. Pour the supernatant fluid (by
completely inverting the tube) into a second container not the original
one. This can be used for biochemical tests to avoid contaminating the
original urine which may need to be cultured (depending on the findings
of the microscopical examination).
3. Remix the sediment by tapping the bottom of the tube. Transfer one
drop of the well-mixed sediment to a slide and cover with cover glass.
• Note: Do not discard the remaining sediment because this may be needed
to prepare a Gram smear if WBCs and, or, bacteria are seen in the wet
preparation.
– Examine the preparation microscopically using the 10Xand 40X objective with the
condenser iris closed sufficiently to give good contrast.

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 Report on urine wet mount preparation

• Report the following

– Bacteria- report only when freshly passed
• Usually seen as rods, but sometimes cocci or
streptococci
• Bacteriuria is usually accompanied by pyuria (pus cells
in urine
• Note: In a urinary infection, protein and nitrite are
often found in the urine. With E. coli infections, the
urine is markedly acid. An alkaline urine is found with
Proteus infections

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 Wet mount…. Cont’

– White cells (pus cells): These are round, 10–15µm

in diameter, cells that contain granules
• In urinary infections they are often found in clumps. In
urine sediments, white blood cells (WBC) are usually
reported as:
– Few: Up to 10 WBCs/HPF (high power field, i.e. using 40X
objective)
– Moderate number: 11–40/HPF
– Many: More than 40 WBC/HPF

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 Wet mount…. Cont’

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 Wet mount…. Cont’

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 Wet mount…. Cont’

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 Wet mount…. Cont’

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 Wet mount…. Cont’

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 Wet mount…. Cont’
• Red cells: These are smaller and more refractile than white
cells. They have a definite outline and contain no granules.
When the urine is isotonic, they have a ringed appearance
• They are usually reported as few, moderate or many in
number per high power field.
– Note: When the urine is hypertonic, i.e. more concentrated than
the fluid inside the red cells, fluid will be drawn out of the cells
and they will appear smaller than normal and often crenated
(spiky)
– When haematuria is due to glomerulonephritis, the red cells
often vary in size and shape (dymorphic).
– In sickle cell disease, sickled red cells can sometimes be seen in
the urine.

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 Wet mount…. Cont’

• Haematuria (red cells in urine) may be found in

urinary schistosomiasis (usually with proteinuria),
bacterial infections, acute glomerulonephritis
(inflammation of the glomeruli of the kidneys), sickle
cell disease, leptospirosis, infective endocarditis,
calculi (stones) in the urinary tract, malignancy of the
urinary tract, and haemorrhagic conditions.
– Note: The finding of red cells in the urine of women may
be due to menstruation.

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 Wet mount…. Cont’

• Casts: These can usually be seen with the 10X

objective provided the condenser iris is closed
sufficiently to give good contrast.
– They consist of solidified protein and are cylindrical in
shape because they are formed in the kidney tubules.
– The following casts can be found in urine:
– Hyaline casts, which are colourless and empty
• They are associated with damage to the glomerular filter
membrane.
• A few may be seen following strenuous exercise or during fever.

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 Wet mount…. Cont’

• Waxy casts, which are hyaline casts that have remained

in the kidney tubules a long time.
– They are thicker and denser than hyaline casts,
often appear indented or twisted, and may be
yellow in colour.
– They usually indicate tubular damage and can
sometimes be seen in renal failure.

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 Wet mount…. Cont’
• Cellular casts, which contain white cells or red cells.
– Red cell casts appear orange red.
• They indicate haemorrhage into the renal tubules or
glomerular bleeding.
– White cell casts are found when there is inflammation
of the kidney pelvis or tubules.
– Yellow-brown pigmented casts may be seen in the
urine of jaundiced patients.
• Granular casts, which contain irregular sized granules
originating from degenerate cells and protein.
– They are also associated with renal damage.

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 Wet mount…. Cont’
• Yeast cells: These can be differentiated from red cells by
their oval shape and some of the yeasts usually show single
budding.
– If in doubt, run a drop of dilute acetic acid under the cover glass.
Red cells will be haemolyzed by the acid, but not yeast cells.
– Note: Glove powder in urine also resembles yeasts.
• It can be distinguished by adding a drop of iodine (as used in Gram
stain). Glove powder granules (starch), turn blue-black.
– Yeast cells are usually reported as few, moderate, or many per
HPF.
– They can be seen in the urine of women with vaginal
candidiasis, and occasionally in specimens from diabetics and
those with immuno-suppression.

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 Wet mount…. Cont’

– Epithelial cells: These are easily seen with the 10X

objective.
• They are nucleated and vary in size and shape.
• They are usually reported as few, moderate, or many in
number per low power (10X objective) field.
• It is normal to find a few epithelial cells in urine.
• When seen in large numbers, however, they usually
indicate inflammation of the urinary tract or vaginal
contamination of the specimen.

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 Wet mount…. Cont’

• Crystals
– These have a characteristic refractile appearance.
– Normal urine contains many chemicals from which
crystals can form, and therefore the finding of
most crystals has little importance.
– Crystals should be looked for in fresh urine when
calculi (stones) in the urinary tract are suspected.
– Crystals which may be found in rare disorders
include:

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 Crystals…. Cont’
• Cystine crystals, which are recognized by their
six-sides.
– They are soluble in 30% v/v hydrochloric acid
(unlike uric acid crystals which they may
resemble).
– They can be found in cystinuria, a rare congenital
metabolic disorder in which cystine is excreted in
the urine.

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 Wet mount…. Cont’

• Cholesterol crystals, which look like rectangles

with cut-out corners.
– They are insoluble in acids and alkalis but soluble in
ether, ethanol, and chloroform.
– They are rarely found except in severe kidney disease
or when a lymphatic vessel has ruptured into the
renal pelvis.
• Tyrosine crystals, which are yellow or dark-
coloured and look like needles massed together.
– They are insoluble in ethanol, ether, and acetone.
– They are occasionally found in severe liver disease.

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 Wet mount…. Cont’

• Other crystals found in urine

– Occasionally sulphonamide crystals are found in the urine
of patients being treated with sulphonamides.
– When deposited in the urinary tract they can cause
haematuria and other complications.
– Triple phosphate crystals are occasionally found in alkaline
urine. They have little or no clinical significance.
– Calcium oxalate crystals are frequently seen. When found
in freshly passed urine they may indicate calculi in the
urinary tract.
– Uric acid crystals are yellow or pink-brown. They can
sometimes be found with calculi

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 Crystals … cont’

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 Day 1… cont’

3. Test the specimen biochemically

• Biochemical tests which are helpful in
investigating UTI include:
– Protein
– Nitrite
– Leukocyte esterase

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 Day 1… cont’
4. Culture the specimen
• It is not necessary to culture urine which is microscopically
and biochemically normal, except when screening for
asymptomatic bacteriuria in pregnancy.
• Culture is required when the urine contains bacteria (as
indicated by the Gram smear), cells, casts, protein, nitrite,
or has a markedly alkaline or acid reaction.
• Estimating bacterial numbers
– It is necessary to estimate the approximate number of bacteria
in urine because normal specimens may contain small numbers
of contaminating organisms, usually less than 10 000 (10 4) per
ml of urine.

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 Day 1… cont’

• Urine from a person with an untreated acute urinary infection

usually contains 100 000 (10 5) or more bacteria per ml.
• The approximate number of bacteria per ml of urine, can be
estimated by using a calibrated loop or a measured piece of
filter paper.
• Both methods are based on accepting that a single colony
represents one organism.
• For example, if an inoculum of ml produces 20 colonies, the
number of organisms represented in 1/500 ml of urine is 20,
or 10 000 in 1 ml (500 X 20).

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 Day 1… cont’
• The calibrated loop method using quarter plates of culture
media is recommended because it is inexpensive, simple to
perform, and provides individual colonies that are easier to
identify and remove for antimicrobial susceptibility testing.
• Cystine lactose electrolyte-deficient (CLED) agar
– Mix the urine (freshly collected clean-catch specimen)
by rotating the container.
– Using a sterile calibrated wire loop, e.g. one that
holds ml (0.002 ml), inoculate a loopful of urine on a
quarter plate of CLED agar. If microscopy shows many
bacteria, use a half plate of medium.
– Incubate the plate aerobically at 35–37 °C overnight.

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 Day 2 and onwards
4. Examine and report the cultures CLED agar
culture
– Look especially for colonies that could be:
• Escherichia coli (perform indole and beta-gluca-ronidase
tests for rapid identification
• Proteus species
• Pseudomonas aeruginosa
• Klebsiella strains
• Staphylococcus aureus
• Staphylococcus saprophyticus
• Enterococcus faecalis

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 Day 2 and onwards…. Cont’
• Reporting bacterial numbers
– Count the approximate number of colonies.
– Estimate the number of bacteria, i.e. colony-forming units (CFU)
per ml of urine. Report the bacterial count as:
• Less than 10 000 organisms/ml (104/ml), not significant.
• 10 000–100 000/ml (104–105/ml), doubtful significance (suggest
repeat specimen)
• More than 100 000/ml (105/ml), significant bacteriuria.
• Example
– If 25 E. coli colonies are counted and a 1/500 ml loop was used,
the approximate number of CFU per ml of urine: 500 X 25=12
500
– Such a count would be reported as:
• 10 000–100 000 E. coli/ml

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 Day 2 and onwards … cont’

• Antimicrobial susceptibility testing

– Perform susceptibility testing on urines with signifi-cant
bacteriuria, particularly from patients with a history of
recurring UTI.
– Cultures from patients with a primary uncomplicated
UTI may not require a susceptibility test

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