Microbiological examination of urine sample

1. Microbiological examination of
urine sample
Saajida Sultaana Mahusook

2. • Urine is the fluid produced by the kidneys that
carries water and wastes through the urinary tract
and then is eliminated from the body.
• Normally, urine in the bladder and the urinary
tract from kidney to the last third of the urethra is
sterile.
• When the urinary tract becomes infected with
bacteria, an inflammation at the site of infection
results in symptoms of urinary tract infection
(UTI), and significant numbers of bacteria and
white blood cells (WBC) appear in the urine.
• The urine culture is a test that detects and
identifies bacteria and yeast in the urine, which
may be causing a urinary tract infection (UTI).

3. Symptoms of a UTI
• A strong, persistent urge to urinate.
• A burning sensation when urinating (painful).
• Passing frequent, small amounts of urine.
• Urine that appears cloudy.
• Urine that appears red, bright pink or cola-
colored — a sign of blood in the urine.

4. Sample Requirement
• The genital area is cleaned before collecting your
urine
• The mid-stream clean catch urine sample is the
most common type of sample collected.
• Urine may also be collected using a catheter and,
rarely, a needle is used to aspirate urine directly
from the bladder.
• For infants, a collection bag may be attached to
the genital area to catch any urine produced.
• Urine samples not cultured immediately
following collection were refrigerated at 4°C until
processing.

5. TESTS
Drop method
• After homogenization of the urine sample, a hanging drop of approximately 50 μl
of the urine was deposited, on microscope slide , allowed to dry, without spreading,
at ambient temperature.
Loop method
• A 10-μl volume of homogenized urine sample was applied, by means of a nickel-
chrome loop calibrated on microscope slide and was allowed to dry, without
spreading, at ambient temperature.
Gram staining
After air drying, the smears were fixed by passing the slides two or three times through
the flame of a Bunsen burner, and then they were stained by Gram method.
Microscopic observation
• with a 100× oil immersion objective, 50 fields were examined, and the shapes and
number of microorganisms and cells per field were recorded.
• The microscopic reading was done systematically, beginning at the edge of the
central region of the smear and continuing across its diameter.
• A positive microscopic examination was defined as the presence of ≥2
microorganisms uniformly distributed per oil immersion field, after observation of
at least 20 fields, according to the criteria of Washington et al.

6. Microscopic field (oil immersion objective) showing the accumulation of E. coli at the
edge of the urine drop (to the left) with the Gram-stained preparation of 10 μl of a
positive urine specimen (≥105 CFU/ml). A DIAPLAN microscope (Leitz, Wetzlar,
Germany) and Tri-X pan ISO 400/27 film (Eastman Kodak Company, Rochester, N.Y.)
were used.

7. Semiquantitative urine culture
• Semiquantitative urine culture using the calibrated loop technique
and inoculation on cystine-lactose-electrolyte-deficient agar (Difco
Laboratories, Detroit, Mich.) was employed as the reference
method.
• Samples were considered positive if they contained ≥105, or 104 to
<105, CFU of the urinary pathogen/ml of pure culture.
• Also considered to indicate a urinary infection was isolation of two
potentially pathogenic bacterial species, when the individual counts
for the two species were >105 and >105, >105 and >104, or >104 and
>104 CFU/ml, or when the count for one organism was
>104 CFU/ml and it was clearly predominant, i.e., at least 10-fold
more than the other.
• Urine specimens containing ≥105 or <105CFU of nonpathogenic
bacteria (lactobacilli, diphtheroids,Staphylococcus epidermidis, or
non-group-D Streptococcus spp.)/ml or multiple (three or more)
species of gram-negative bacteria, obtained from patients without
clinical evidence of urinary infection, were considered contaminated
and were excluded from the study.
• Isolated microorganisms were identified by standard biochemical
procedures.

8. The urine culture is used, to diagnose a urinary tract infection
(UTI) and to identify the bacteria or yeast causing the infection.
If a urine culture is positive, susceptibility testing may be done to
determine which antibiotics will inhibit the growth of the microbe
causing the infection. The results will help a healthcare
practitioner determine which drugs are likely to be most effective
in treating your infection.
A urine culture is used, to screen pregnant women for
asymptomatic bacteriuria, a condition in which significant
amounts of bacteria are in the urine but do not cause symptoms.
About 2%-10% of pregnant women in the U.S. have this
condition that can lead to more serious kidney infection as well as
increased risk of preterm delivery and low birth weight.

9. FINDING OTHER INFECTIONS
• Urine cultures can detect some sexually transmitted diseases
but not the test of choice for sexually transmitted diseases in
adults. Urine cultures may be used to test for STDs in
children.
• Some STDs such as chlamydia may be tested using a urine
sample, but the testing method used detects chlamydia
genetic material in the urine and is not a culture.
• For another example, a urine culture may be used to help
diagnose infections of the urinary tract and genital tract
caused by mycobacteria. Typically, this test requires that the
first urine voided in the morning be collected.

10. • CREDITS
• https://labtestsonline.org/tests/urine-culture
• https://pubmed.ncbi.nlm.nih.gov/?term=%22S
pecimen+Handling%22%5BMeSH%5D
• https://jcm.asm.org/content/36/3/820
• www.mayoclinic.org
THANK YOU