EXAMINATION OF URINE SPECIMEN FROM INFECTED URINARY

TRACT REGION

Urinary tract is normally sterile except for the urethra, which may
contain few commensals like Acinobacter (Acetic acid bacteria). The
presence of microbes occurs usually as a result of infection. The presence of
bacteria in urine is known a BACTERIURIA. Hence, the diagnosis is
urinary tract infection is always made with bacteriological examination of
the urine.

Materials:
- Sterile urine collection bottle
- Blood agar medium
- CLED medium
- MacConkey medium or any other suitable needs
- Inoculating loop
- Centrifuge

Collection of Sample:
During collection, the urine may become contaminated with skin
commensals including those from vagina in case of females.
It is very important that the urine specimen be collected with little or no
contamination, by collecting at mid stream during urination. This is called
CLEAN-CATCH specimen. The urine specimen is usually colled in sterile,
wide mouthed, leak proof container and transported to the laboratory without
delay (within a period not later than 2 hours). This is because urine is a rich
culture medium for rapid bacteria growth, so delay of urine for more than 2
 hours gives room for bacteria multiplication and this will give unreliable
count. This can be prevented by storage in a refrigerator or by transporting
the specimen in a special container that maintains low temperature
especially if the health centre is far away to the microbiological laboratory.
The addition of boric acid powder (0.1g/10ml of urine) also helps to
preserve the urine prior to its culturing.
For bacteriological examination, midstream urine is usually collected
except is tuberculosis of the urinary tract is suspected. In such case, the first
early morning urine passed in the day is most suitable and this should be
collected for three consecutive days for proper tests. For female patients, the
urine is passed out with the labia separated and the middle of the stream
collected. This helps to reduce rate of contamination by the commensal.

PROCEDURE FOR THE EXAMINATION OF URINE SPECIMEN

1. Macroscopic Examination:
This involves noting the appearance of the specimen (urine). Normal freshly
passed urine is pale yellow or yellow in terms of colour and clear.

2. Testing the Specimen (urine) Biochemically using Strip:

This indicates the presence of protein, glucose, bilirubin, ketones etc.

3. Microscopic Examination:
Microscopic examination using freshly centrifuged urine is carried out by
placing few drops of well-mixed centrifuged urine on a slide, cover with a
cover slip and examine under the microscope using x10 and x40 objectives.
The presence of bacteria indicates urinary tract infection. Pus cells are
usually present.
4. Culturing of the Specimen:
Culture is necessary if the urine has been found to contain bacteria, other
cells, protein etc. The media used for routine analysis are mostly blood agar
Blood agar medium is used because it helps in rapid identification of
haemolytic bacteria that grow poorly or not at all on MacConkey or CLED
agar.
The culture method usually adopted is semi-quantitative instead of
quantitative method which is time consuming and expensive. The two semi-
quantitative methods in use are loop of standard diameter is used and this is
charged with well mixed centrifuged urine and inoculated into a plate of
solid medium in a standard form.
After overnight incubation at 35-37oc, the number of colonies developed in
the plate are counted. If bacteria count of 105 or more is observed, it means
that there is UTI an d thus the count is significant. A count of 10 4-105 could
be regarded as either infection or contamination. A much lower count (i.e.0
<104) indicates contamination and not infection unless if the urine sample
was from a patient on antibiotic, in such a case, is regarded as significant.
Possible bacteria pathogens include E. coli, Proteus spp, Pseudomonas
aeroginosa, haemolytic Streptococcus, Staphhylococcus saprophyticus,
Klebsiella spp and Enterococcus spp.