Examination of Urine

By: Mazen Mohammed Qaid

 Possible Pathogenic Bacteria
• Gram positive • Gram negative
1. Staphylococcus 1. Escherichia coli(UPEC)
saprophyticus 2. Proteus species
2. Haemolytic streptococci 3. Pseudomonas aeruginosa
4. Klebsiella strains
* These species are not primarily 5. Salmonella Typhi*
pathogens of the urinary tract, but 6. Salmonella Paratyphi*
may be found in urine. 7. Neisseria gonorrhoeae *

-Also Mycobacterium tuberculosis, Leptospira interrogans,

Chlamydia, Mycoplasma and Candida species.
 Commensals
• The bladder and urinary tract are normally sterile.
• The urethra and perineum has wide variety of Gram
positive and negative organisms which can
contaminate urine when it is being collected. With
female patients, the urine may become contaminated
with organisms from the vagina.
• Most urine specimens will contain fewer than 10000
contaminating organisms per ml.
Types of specimen

• Midstream clean catch urine

• Suprapubic aspiration

• Catheterized urine.
Criteria of specimen rejection

• Un-refrigerated specimen older than 2 hours.

• Unlabeled specimen.
• Mislabeled specimen.
• Specimen in expired transport container.
• 24 hours urine specimens.
Collection and transport
• Stop antibiotics (for 3 days)
• 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 collected
in sterile, dry, wide-necked, leak-proof container.
• Request 10–20 ml urine.
• Tell the patient not to touch the inside or rim of the container.
• Deliver to the laboratory immediately.
• The maximum time allowed for processing is 2 hours from the
collection.
Continue,
• If a delay longer than 2 hour, it must be
refrigerated at 4–6 °C or
• add boric acid powder (0.1 g/10 ml of urine) to
preserve the specimen, and mix well.
• Important: Instruct the patient before the
collection to collect the urine with as little
contamination as possible, preferably with
illustration.As following:
Male:
1. Wash the hands before collecting a specimen
2. If not circumcised, draw back the foreskin.
3. Begin to urinate, but pass the first portion into
the toilet.
4. Collect the mid-portion of urine into the
container, and pass the excess into the toilet.
Female:
1. Wash the hands.
2. Cleanse the area around the urethral opening
with clean water, dry the area with a sterile gauze
pad.
3. Separate the labia with one hand.
4. Void the first portion of urine into the toilet.
5. Collect the mid-portion of urine into the
container and pass the excess into the toilet.
Infants:
Have ready: Clean, preferably sterile container of
appropriate size or a plastic bag, cotton wool or gauze
pads, handwarm soapy water.
1. Clean the external genitals.
2. Give the child as much liquid as possible just prior to
the collection.
3. Seat the child on the lap of the mother, nurse or ward
attendant.
4. Collect as much urine as possible in the container or
plastic bag when the child urinates.
Laboratory examination

Day 1
1. Macroscopic examinations
Describe the appearance
Urinary Chemstrip (dipstick)
2. Microscopic examination: (a drop of urine sediment)
wet preparation
Gram stain: for pus cells and bacteria.
ZN stain : for M. tuberculosis
3.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 markedly alkaline or acidic.
 Mix the urine (uncentrifuged) by rotating the
container.
 Using a sterile calibrated wire loop (1 or 10 ul)
inoculate a loopful of urine on plate of CLED.
Incubate aerobically at 35–37 °C overnight.
 Cystine lactose electrolyte-deficient (CLED) agar

• Is differential, non selective culture medium used for the

isolation, enumeration and differentiation of urinary
microorganisms.
CLED agar is widely used to isolate urinary pathogens;
• Allows the growth of both Gram negative and Gram positive
pathogens.
• Electrolyte-deficient to prevent the swarming of Proteus
species.
• bromothymol blue indicator.
• L-F colonies appear yellow.
Method of colony count
 Day 2
1. Examine and report the cultures.
Appearance of some urinary pathogens on CLED agar
• E. coli: Yellow (lactose-fermenting) opaque colonies often with
slightly deeper colored center
• Klebsiella species: Large mucoid yellow or yellow-white
colonies.
• Proteus species: Translucent blue-grey colonies.
• P. aeruginosa: Green colonies with rough periphery
(characteristic color).
• E. faecalis: Small yellow colonies.
• S. aureus: Deep yellow colonies of uniform color.
• S. saprophyticus and other coagulase negative staphylococci:
Yellow to white colonies
2. Reporting bacterial numbers

• Count the approximate number of colonies.

• Estimate the number of bacteria, i.e. colony-
forming units (CFU) per ml of urine.
 Interpretation of Urine Culture
• Remember! 50-60% of urine cultures received in the laboratory will be “No
growth” or “growth of contaminants"only.
• After 24 Hours of Incubation: No growth on either plate. Reincubate plates
for an additional 24 hours in case there is an organism that grows more
slowly. If visible growth on plate(s) Evaluate the growth.
• If the calibrated loop used was 0.01 ml then each colony equals 100 cfu. -
Example: 13 colonies seen x 100 = 1,300 cfu/ml.
• If the calibrated loop used was 0.001 ml then each colony equals 1000 cfu
- Example: 13 colonies seen x 1000 = 13,000 cfu/ml.
• If the number of colonies of one colony type is more than 100,000, then
report out colony count as >100,000 cfu/ml .Confluent growth of bacteria,
covering most of the surface area of the plates can be read as >100,000
cfu/ml.
• If there is no growth on the plate then report out : Using 0.01 loop: <100
cfu/ml, No growth. If using 0.001 loop : <1000 cfu/ml, No growth
Example
If 25 E. coli colonies are counted and a 1ul (0.001ml)
loop was used, the approximate number of CFU per ml of
urine: 1000 x25= 25000 Such a count would be
reported as: 10 000–100 000 E. coli/ml
3. Staining the bacterial colonies in culture by gram
stain.
4.perform Biochemical test
Catalase, coagulase, Oxidase, Urase, Bile solubility test,
DNase, nitrate, and optochin disc.
5.Antimicrobial susceptibility test.Using Muller Hinton
agar
 Day 3
1. See the biochemical tests results and record the
name of bacteria.
2. Measure the zone of inhibition of antibiotics
and record the report