Collection, transport and examination of urine specimen

Definition: The presence of significant numbers of micro-organisms any where in the urinary tract.
NB: Kidney and bladder are sterile at normal state.
The commonest causative agents of UTI are gram-negative rods.
These are: Escherichia coli
Pseudomonas aeruginosa
Klebsiella pneumoniae
Proteus spp.
Enterobacter aerogens
Other important causative agents: Enterococci
Staphylococcus saprophyticus
Collection of urine
A freshly voided urine specimen is adequate for most urinalysis. The patient should be instructed to void
directly into a clean, dry container, or a clean, dry bedpan so that the specimen can be transferred to an
appropriate container. Specimens from infants and young children can be collected in a disposable
collection apparatus. If a urine specimenn is likely to be contaminated with vaginal discharge or
menstrual blood, this period has to be avoided and the patient must be informed to bring a clean-voided
specimen. All specimens should be immediately covered and taken to the laboratory.
Types of Specimen
First Morning Specimen - a speciemen obtained during the first urination of the day.
􀂃 Most concentrated
􀂃 Bladder incubated
Best for:
􀂃 Nitrite
􀂃 Protein
􀂃 Microscopic examination
Random Specimen - a spciemen obtained at any time during examination.
􀂃 Most convenient
􀂃 Most common
Good for:
􀂃 Chemical Screen
􀂃 Microscopic examination
Second-voided Specimen - In this case first morning specimen is discarded and the second specimen is
collected and tested. Such type of specimen is good for:
􀂃 Reflection of blood glucose.
􀂃 Keeping of formed elements intact.
Postprandial : a specimen obtained 2 hours after meal.
􀂃 Good for glucose
24- Hour specimen - a specimen obtained within 24 hours.
􀂃 Necessary for quantitative tests, especially for quantitative determination of protein.
Procedure for Collection of 24 hour Urine Specimen
1. Inform or Direct the patient to completely empty his bladder and discard his urine exactly at the
beginning of the 24 hour time collection (let say at 6:00 a.m.).
2. Collect all urine voided during the following 24 hours, including that voided exactly at the end of the
24 hour period in a container (at 6:00 a.m.) of the following (second) day.
3. All the urine collected must be preserved.
4. The container should be labeled with :
􀂃 The test order
􀂃 The patient’s name
􀂃 Time of collection
􀂃 The preservative added
Mid- stream Specimen - a specimen obtained from the middle part of the first urine.
􀂃 It is commonly used for routine urinalysis.
􀂃 It is also important for bacteriological urine culture.
Clean Catch Urine Specimen
Used for microbial culture and routine urinalysis. When specimens are collected for bacteriological
examination they should be collected by the ‘clean catch’ method or by catheterization into sterilized
container.
Catheterization is the process of passing a tube through the urethra to the bladder for the withdrawal of
urine (it may introduce urinary tract infection).
The best method is properly collected ‘clean catch’ urine, which is collected as follows :
a. The genital area should be cleaned with soap and water and rinsed well. This is to keep off bacteria on
the skin from contaminating the urine specimen.
b. The patient should urinate a small amount and this is discarded.
c. The urine that comes next, the mid-stream specimen, should be collected into a sterile container of 30
to 50 ml.
d. After obtaining the specimen the patient continues to urinate and this is discarded.
Sources of Errors in the Collection of Urine
1. Bacteriologically or chemically contaminated specimen.
2. Wrong type/amount of preservative.
3. Partial loss of specimen or inclusion of two-morning specimen in
the 24 hr collection.
4. Inadequate mixing of specimen before examination.
5. Careless measuring of the 24 hr volume.
2.2 Preservation of Urine Specimen
Urine should be examined immeditely as much as possible after it is passed, because some urinary
components are unstable. If urine specimen can not be examined immediately, it must be refrigerated or
preserved by using different chemical presevatives. The maximum time that urinary contents to be
maintained in urine specimen is one hour.Long standing of urine at room temperature can cause :
􀂃 Growth of bacteia
􀂃 Break down of urea to ammonia by bacteria leading to an increase in the pH of the urine and this may
cause the precipitation of calcium and phosphates.
􀂃 Oxidation of urobilingen to urobilin
􀂃 Distruction of glucose by bacteria.
􀂃 Lysis of RBCs, WBCs and casts.
Laboratory diagnosis
Specimen: Clean catched midstream urine
Catheterized urine
Suprapubic aspiration
Direct microscopic examination: WBCs, RBCs, Epithelial cells.
The presence of more than five WBCs and abundant epithelial cells per HPF supports infection of urinary
tract.
Gram stain: The presence of one bacterium in Uncentrifuged gram stained urine confirms Urinary tract
infection.
Culture: Blood agar medium, Mac Conkey agar medium
Interpretation of culture results
1. ≥105cfu/ml of urine is significant to indicate UTI.
2. <103cfu/ml of urine indicates contamination of specimen.
3. 103-105cfu/ml of urine is uncertain.
NB: 103-105cfu/ml of urine in symptomatic patient or suprapubic or catheterized specimen indicates
UTI.

Collection, Transport and Examination of urogenital specimens possible pathogens

Urethral swabs
• N. gonorrhoeae
• S. Pyogenes
• Ureaplasma urealyticum
• Chlamydia trachomatis and
• Occassionally Trichomonas vaginalis
Cervical swabs from non-puerperal women:
• N. gonorrhoeae
• S. pyogenes
• Other B.hemolytic streptococci
• Chalmydia trachomatis and
• herpes simplex virus
Cervical swabs from women with puerperal sepsis or septic abortion:
• S. pyogenes
• Other B – haemolytic
• Streptococci
• Anaerobic streptococci
• Enterococci
• S. aureus
• Clostridium perfringens
• Listeria monocytogenes
• Bacterioles species
• protens species
• E. Coli & other coliform

Vaginal Swabs:
T. vaginalis
Candida species
Gardnerella vaginalis (Haemophilus vaginalis)
Neisseria gonorrhoeae.

Fluid and pus from genital ulcers

T. pallidium
C. trachomatis
Calymmatobacterium granulomatis (Donovania granulomatis) (Klebsiella granulomatis)
H. ducreyi.

Collection and transport of urogenital specimen

Amies medium is the most efficient medium for transporting urethral, cervical and vaginal swab
Specimen required for diagnosis of gonorrhoea
Male patients:
. Smears of urethral discharge
. Rectal swab from homosexual patient
Female patients
. Smears of mucopus from the cervix and urethra
Note: N. gonorrhoeae infects the mucous membranes of the cervix, not the vagina.
The pathogen is, therefore, more likely to be isolated from a cervical swab than from a vaginal swab.

Collection of Urethral specimens

1. Clean the urethral opening using a swab moistened with sterile physiological saline.
2. Gently massage the urethra from above downwards, and collect a sample of pus on a sterile cotton
wool swab.
• The patient should not have passed urine preferably for 2hours before the specimen is collected.
3. Make a smear of the discharge on a slide for staining by the Gram technique and label the specimen.

Collection of Cervical specimen

1. Moisten a vaginal specimen with sterile warm water and insert into the vagina.
2. Cleanse the cervix using a swab moistened with sterile physiological saline.
3. Pass a sterile cotton wool swab into the endocervical canal and gently rotate the swab to obtain a
specimen
• Amies transport
• Make a smear of the cervical mucopus for Gram stainingl and lable the specimen
Collection of vaginal specimen
• Using sterile swab, collect a sample of vaginal discharge.
- Amies
- Smear and label the sample

Laboratory examination of urogenital specimen

Routine culture
• MNYC medium
Incubate in moist CO2 atmosphere
⇒Neisseria gonorroeae
Microscopy
⇒Gram smear:
Look for pus cells and bacteria
Suspected gonorrhoeae:
Look for intracellular gram negative diplococci

vaginitis:
Look for yeast cells, and epithelial cells in the gram variable coccobacili

Suspected puerperal sepsis or septic abortion

Look for gram positive rods, streptococci, cocci and gram negative rods.

Suspected chanchroid
Look for Gram negative coccobacilli showing bipolar staining

Additional culture
Blood agar (aerobic and anaerobic), macCokey agar,and cooked meat medium, if puerperal sepsis
or septic abortion is suspected
Sabourand medium, if vaginal candidiasis is suspected and yeast cell not detected microscopically
Serum culture, if chancroid is suspected ⇒H. ducreyi
Microscopy
Saline preparation, if trichomoniasis is suspected.
Giemsa stained smear: If donovanosis is suspected
Dark field preparation, if syphilis is suspected.
Collection, transport and examination of skin specimen
Possible pathogens
Gram positive Gram negative
S. aureus Escherichia coli
S. pyogenes Proteus
Enterococci Pseudomonas aeruginosa
Anaerobic streptococci yersinia pestis
Bacillus anthracis Vincent’s organisms
Corynebacterium ulcerans
Also M. leprae Virus: pox viruses and herpes viviruses
M. ulcerans Fungi: Ringworm
T. Pertenue parasite: Leishmania spps
T. Carateum : onchocerca volvulus
:D. medinensis

Commensals
Gram positive Gram negative
Staphylococci Escherichia Coli and other coliforms
Micrococci
Anaerobic cocci
Viridans streptococci
Enterococci
Dephtheoids

Collection of skin specimens and ulcer materials

1. Using a sterile dry cotton wool swab, collect a sample of discharge from the infected tissue.
If there is no discharge, use swab moistened with sterile physiological saline to collect a specimen.
Insert the swab in a sterile tube.
􀂾 If the tissue is deeply ulcerated and necrotic (full of dead cells); Aspirate a sample of infected material
from the side wall of the ulcer using a sterile needle and syringe.
􀂾 Fluid from pustules and blisters: Aspirates a specimen using a sterile needle and syringe.
􀂾 Serous fluid from skin ulcers, papillomas or papules, that may contain treponemes:
• Collect a drop of the exudates directly on a clean cover glass and invert on a clean slide.
• Delivery immediately the speciemen to the laboratory for examination by darkfield microscopy.
2. If the specimen has been aspirated, transport the needle and syringe in a sealed water proof container
immediately to the laboratory.

Laboratory examination of skin specimens

1) Culture the specimen
Blood agar and MacConkey
• Inoculate the specimen
• Incubate both plate aerobically at 35-370C overnight.
Additional:
Sabourand agar if a fungal infection is suspected
• Inoculate to agar plate
• Send to a Mycology Reference laboratory.
Modified Tinsdale Medium (MTM), if cutaneous diphitheria is suspected:
• Inoculate the sample for isolation of C. Ulcerans
• Incubate aerobically at 35-370C for up to 48hours, examining the growth after overnight incubation.
Blood agar and MacConkey agar at room temperature, if bubonic plague is suspected:
• Inoculate the specimen
• Incubate both plates aerobically at room temperature for upto 48hours.
• Examination for growth after overnight incubation.
􀀹 Y. pestis is a highly infectious organism.
Maximum care should be taken
Lownstein Jensen (LJ) Meduim if Buruli ulcer is suspected:
• Decontaminate the swab by immersing it in sodium hydroxide 40g/l (4% W/v) solution for 10 minutes.
• Inoculate the decontaminated specimen on two slopes of acid LJ medium.
• Incubate one slope at 35-370C as described for culture of M. tuberculosis and incubate the other slope
at 320C for up to 8 weeks.
2.) Microscopical examination
Routine:
Gram Smear:
Look for:
• Gram positive cocci that could be S. aureus
• Gram positive streptococci that could be S. pyogenes or other streptococci.
• Gram negative rods that could be p. aeroginosa, proteus
speices, E.coli or other coliforms.
􀀹 If tropical ulcer is suspected, look for vincent’s organisms
􀀹 If cutaneus anthrax is suspected, look for large Gram variable rods lying in chains that could be B.
anthracis.
• Examine also a polychrome methylene blue stained smear.
Additional:
Potassium hydroxide preparation, if ringworm or other superficial fungi infection is suspected.
For detection of ringworm:
Polychrome Loefler methylene blue smear if cutaneous anthrax is suspected.
- Fix with potassium permanganates 40g/l solutions for 10 minutes.
- Look for chain of large blue-stained capsules characteristic of B. anthracis.
Ziel-Neelsenstained smear if buruli ulcer is suspected examine for acid fast bacilli.
Dark-field microscope to detect treponemes
- look for motile treponeme if yaws or pinta is suspected
Examine and report the culture
Blood agar and MacConkey agar cultures
Look for: S. aureu, Enterococci, Proteus species, Escherichia coli
S. pyogenes
P. aeniginosa
sabouraud agar -if fungal infection is suspect
Lowenstein Jensen -if Buruli ulcer (caused by mycobacterium ulcerans) is suspected
MTM- if cutaneous diphtheria is suspected