MICROBIOLOGY OF

GENITOURINARY TRACT
LAB ACTIVITY GUS
Julia Hartati, dr.

Learning Objective
Understand methods of specimen collection and lab
exam to confirm the diagnosis UTI and STI.
Collection of Urine Samples for
Culture

Midstream urine specimen

Other voided urine specimens

Catheter collections

Suprapubic aspiration
 Midstream Urine Specimens
1. Wash your hands well with soap and water for
30 seconds. Open the package of disposable
wipes that the nurse gave you, and place it on
a clean, dry, nearby surface
 Midstream Urine Specimens
2. Remove the lid from the specimen cup
and place it flat side down. Do not touch
the inside of the cup or lid.
 Midstream Urine Specimens
3. Sit as far back on the toilet as you can
Spread your labia apart with one hand,
keeping the folds separated
 Midstream Urine Specimens
4. Using the wipes, clean the area between
the labia and around the urethra well from
front to back. Use a new wipe for each
stroke.
 Midstream Urine Specimens
5. Urinate a small amount into the toilet.
After 1 or 2 seconds, hold the cup below
your urine stream and catch about 1 ounce
(30 ml) of urine in the cup. Don’t allow the
cup to touch your skin at any time.
 Midstream Urine Specimens
6. Place the lid on the cup and return it to the
nurse.

• Note: Don’t drink a lot of water before the

test. This could affect the test results.

• The accuracy of the urine collection procedure

can be monitored over time by noting the
frequency with which urine colony counts range
between 10.000 and 100.000 CFU/mL.
 Midstream Urine Specimens
• Specimens should be processed within 2
hours after collection to achieve accurate
colony counts.

• The B-D Urine Collection Kit (Becton-

Dickinson, Cockeysville, MD), designed to
maintain the bacterial population in urine at
room temperature for 24 hours, has been
equally as effective as overnight refrigeration
of specimens.
 Other voided urine specimens
• If one is investigating urethritis, however, it is
actually the initial portion of voided urine that is of
interest.
• Alternatively (although more painfully), a swab may
be inserted into the distal urethra to collect the
specimen.
• The primary application of initial voided urine
specimens is the diagnosis of urethritis caused by
Neisseria gonorrhoeae and Chlamydia trachomatis.
• The most common technique is differential culturing
of urine before and after prostatic massage. A 10-
fold increase in bacterial numbers after massage of
the prostate suggest the diagnosis
Other voided urine
specimens
 Catheter Collections
• Catheterization should be restricted to patients who are
unable to produce an adequate midstream sample, and it
should be performed with meticulous attention to aseptic
technique.
• The first several milliliters of urine from the catheter should be
discarded to wash out any organisms that may have lodged in
the catheter tip during transit through the urethra
• Urine samples can be obtained from indwelling catheter using
a no. 28 needle and a syringe.
• Be sure to disinfect the area where the needle puncture is to
be made.
• Urine can be aspirated through the soft rubber connector
between yhe catheter and the collecting tubing. Urine
samples should not be obtained from catheter bags.
Catheter Collections
 Suprapubic Aspiration
• Are reserved almost exclusively for neonates
and small children.
• The procedure is best performed when the
bladder is full.
 Suprapubic Aspiration
1. The suprapubic skin overlying the urinary
bladder is disinfect and sterile drapes are put
in place.

2. In the immediate site where the tap is to be

made, an anesthetic solution, such as 1%
lidocaine HCl, is injected subcutaneously.

3. With the point of a sharply through the

epidermis with an 18 gauge, short-bevel spinal
needle is gently extended into the urinary
bladder and 10 mL of urine is aspirated into
the syringe
 Culture of Urine Specimens
• If Escherichia coli  Eosin Methylene
Blue (EMB) agar because of the
distinctive morphology of E. coli on that
media.

• Inoculation of duplicate plates with both

0.01 mL and 0.001 mL calibrating loops is
performed for comparison of counts as a
quality control check and to facilitate
counting colonies.
calibrating loops
 Culture of Urine Specimens
• After delivering the
inoculum from the
calibrated loop, the
surface of each agar
plate should be
completely streaked
over all quadrants so
that semiquantitative
colony counts can be
performed after
incubation
Culture of Urine Specimens
 Culture of Urine Specimens

• A colony count of 105 CFU/mL or

greater is the criterion most
commonly used to determine
whether identification and
susceptibility testing of an isolate
should be performed.
 Colony Forming Unit (CFU)
A sample was serially diluted as follows:

1. One milliliter from the sample was

transferred to 9 ml of sterile media in tube A
and mixed a 1/10 serial dilution.

2. One milliliter from tube A was transferred to

9 ml of sterile media in tube B and mixed 
another 1/10 serial dilution. The total
dilution up to this point is 1/100.
3. Then 1 ml from tube B was transferred to 9
ml of sterile media in tube C and mixed  a
1/10 serial dilution. The total dilution up to
this point is 1/1000.
4. 0.1 ml from each tube was plated.
• Plates A and B contained too many colonies to count.
Plate C is countable. If plate C contained 43 colonies
we can determine the concentration in the sample.
a. 43 CFU/0.1 ml=430 CFU/ml
b. 430 CFU/ml /1/1000 or 430CFU/ml/1x10-
3=4.30x105 CFU/ml
Colony Forming Unit (CFU)
 Screening Test for Urinary Tract
Infection

Tests for Detection of

Screening test for Microscopic
bacterial bacteriuria by
bacteriuria examination
products culture

Screening tests for pyuria LE activity WBC/HPF

Collection of Genital Specimens
• Collection of urethral specimens from males:
exudate may be expressed from the urethral
orifice by gently milking the penis.

• If material is not readily obtained, the tip of

narrow-diameter cotton, rayon, or Dacron swab
on a plastic or aluminium shaft may be inserted
3 to 4 cm into anterior urethra.
• If a culture for C. Trachomatis is being obtained,
the swab should be rotated 360 degrees to
dislodge some of the epithelial cells.
Collection of
Genital
Specimens
 Collection of Genital Specimens
• Collection of urethral specimens from
females: cervical specimen are
collected with the aid of a speculum
after clearing off the cervical mucus
with a large swab.

• A smaller swab with a plastic shaft and

a Dacron or polyester tip is
recommended for obtaining the
specimen.
• The tip of the swab is inserted a few
millimeters past the cervical os, rotated
firmly to obtain both exudate and cervical
cells, and removed, taking care not to
touch the lateral walls of the vaginal canal.
• Vaginal secretions can be aspirated or
collected with a swab.
• Endometrial specimens are best obtained
by inserting the swab through a narrow-
bone catheter that has been introduced
into the cervical canal.
Collection of Genital Specimens
Collection of Specimens from Genital Ulcers
• Ulcers should be cleaned to remove
surface debris and contaminating
bacteria

• For dafkfield examination or

preparation of smears, a scalpel blade
should be used to abrade the base of
the ulcer and transfer the material onto
a glass slide.
• For culture, either a washing of the
base or a cotton swab may be used.

• If intact vesicles are present, a swab

may be used to collect fluid for Tzanck
preparations or herpes culture.

• Swabs for bacterial culture may be

placed in Amies’ or Stuart’s transport
media.
Collection of Specimens
from Genital Ulcers