This document discusses significant bacteriuria and how it is defined as ≥105 CFU/ml of bacteria in a urine specimen, or ≥100 CFU/ml for catheterized specimens. It outlines methods for urine examination including microscopy, culture, and chemical tests. Microscopy looks for white blood cells and casts. Culture methods include loop, pour plate, and dip-slide techniques to quantify bacteria. Chemical tests detect nitrites and leukocyte esterase. The interpretation of urine cultures depends on factors like specimen type and patient symptoms. Asymptomatic bacteriuria requires treatment in pregnant women to prevent pyelonephritis.

1. Significant Bacteriuria
Presented by
Dr. DK Kalra

2. Definition
Significant Bacteriuria term was intended by Kass in 1957 to
provide a means of differentiating between contamination in the
freshly voided specimen of urine and true urinary infection.
The numbers of bacteria (≥105 CFU/ml) greater than those likely to
result from contamination from the urethral meatus and its environs.
 For urine collected via bladder catheterisation, the threshold is 100
CFU/ml.
The threshold is also 100 CFU/ml for women displaying UTI
symptoms.

3. Definition
Asymptomatic Bacteriuria:
 If bacteria were isolated in quantitative counts of
≥105 CFU/mL in a voided urine specimen from
asymptomatic patients.
 Common in pregnancy, patient with indwelling
catheter, and Diabetes Mellitus.

4. Urinary tract infection (UTI)
Definition:
 urinary tract infections are characterized by the presence
of ≥105 CFUs/ml of a single bacterial species or multiple
organisms in two consecutive urine specimens, properly
collected from a person with symptoms or signs of a UTI.

5. Examination
• Macroscopic - Appearance, Proteinuria
• Microscopic – WBC, RBC, Bacteria
• Chemical Tests – Nitrate Reduction, Leucocyte
Estrase, Glucose Oxidation tests
• Culture Method – CLED & MacConkey Media
• Quantitative Tests – Miles and Misra Method,
Pour Plate Method
• Semi Quantitative Tests – Standard Loop, Filter
Paper, and Dip-Slide Tests.

6. Urine microscopy
 Normal- <0-2 pus cells/hpf in males, <0-5/hpf in
females.
 UTI- Pus cells >10/μl.
 Pyelonephritis- White blood cells + white cell casts.
 Transitional epithelial cells- after catheterisation &
transitional cell carcinoma.
 Presence of large number of squamous cells in urine-
contamination with vaginal fluid.

7. Cultural methods
• Loop:
 Double loop used
 Loop size of 4mm which carries 0.01 ml of urine
 Loopful of urine is spread over the surface of the agar
plate by making primary well (semiquantitative purpose)
& secondary well (separate colonies)
• Media used:
 CLED and MacConkey

8. Chemical Tests for Significant
Bacteriuria
Griess Nitrite Test:
 Nitrites are not present in normal urine
 Ingested nitrites nitrates and excreted in urine
 If gram negative bacteria are present in urine with
enzyme nitrate reductase converts nitrates to nitrites
 A typical commercial Griess reagent contains 0.2%
naphthylethylenediamine dihydrochloride, and
2% sulphanilamide in 5% phosphoric acid

9. Griess Nitrite Test…
• If Nitrites are present in urine they will give pink
colour.
• Para-arsanilic acid or sulphanilamide +
NO2 →Diazonium salt
In an acid medium
• Diazonium salt + tetrahydrobenzoquinoline → Pink
azo dye

10. Griess Nitrite Test…
 Some organisms like Staphylococcus, Enterococci do not reduce
nitrate to nitrite.
 Also urine must be retained in the bladder for minimum 4hrs for
conversion of nitrates to nitrites so early morning sample is preferred.
 The nitrite test has a 92% to 100% sensitivity for UTI but only a 35%
to 85% specificity.
 Nitrite test is especially useful in patients with indwelling urinary
catheters to determine whether they are infected or not.

11. Leukocyte Esterase Test
Leukocyte esterase test: (Combur-Test UX strips - Roche)
 It detects esterase enzyme released in urine from granules of
leukocytes, Positive in pyuria.
 If this test is positive urine culture should be done.
 75% to 96% sensitivity and a 94% to 98% specificity for detecting
pyuria. False-positive tests are usually caused by contamination,
often by vaginal secretions. False-negative test can be caused by
hypertonic urine.

12. Chemical Tests…
Reaction catalysed by leukocyte esterase
 Indolecarboxylic acid ester → Indoxyl + Acid
In acid medium
 Indoxyl + Diazonium salt → Violet azole dye
Glucose Test Paper: enzymes used are glucose oxidase and
peroxidase.
 Normal value: 160-180 mg/dl.
 Catalysed by glucose oxidase
 Glucose + O2 → D-glucono-δ-lactone + H2O2
Catalysed by peroxidase
 H2O2 + Chromogen → Oxidised chromogen (coloured) + H2O

13. Quantitative methods:
Miles & Misra Method
Miles & Misra method: (1938)
It is used to determine the CFU in bacterial
suspension or in urine.
Materials required:
 A calibrated dropping pipette delivering drops of 20μl.
 Petri dishes containing nutrient agar or other appropriate
medium.
 Phosphate Buffered Saline (PBS) or other appropriate
diluent.
 Bacterial suspension or homogenate.

14. Quantitative methods
• CFU per ml = Average number of colonies for a dilution
X 50 X dilution factor.
• Advantages:
• Faster than other methods.
• Less bacterial contamination.

15. Pour Plate Method
• In pour plate method bacterial counts are performed
using 8-10 tubes and dilutions up to 108 or 1010 are
made.
• One millilitre of the diluted urine was mixed with
nutrient agar 10-15 ml melted and cooled to 50
centigrade, incubated at 37°C. and examined after 24,
48, and 72 hours.
• Colony count is multiplied by dilution factor and will
give viable count/ml of bacteria.

16. Other Methods
• Roll Tube Method – 2ml nutrient agar melted,
cooled to 50 centigrade, with 0.02 ml pipette one
drop is added to two tubes.
Tubes rolled in horizontal position under cold water
tap to make uniform agar layer, incubated and
colonies counted.
• Surface Method - Various dilutions of specimen,
drop with 0.02 ml pipette on surface from height less
than ½” and incubate for 24 hrs and count colonies.

17. Semi quantitative methods
• Standard loop method:
0.001 ml loopful of urine yields 100 colonies.
The count/ml will be 105 (significant bacteriuria).
• Filter paper method: (Leigh & Williams 1964).
 Count of 25 colonies of bacilli or 30 colonies of cocci
approximately corresponds to 105 bacteria/ml.
• Dip-slide method: With a slide coated with CLED or
MacConkey medium, good for peripheral hospitals.

18. Standard Loop Method
• A 4mm loop would pick up .01 ml of a liquid. 0.001 ml of
urine if yields 100 colonies, then the count per ml will be
105 (significant).
• Can be tested and standardized by diluting an overnight
culture of E.coli (109 org/ml) to 105 org/ml, 104 org/ml
and 103 org/ml and culturing on CLED agar.
• Colonies are counted for each dilution and multiply by
dilution factor (50).

19. Filter Paper Method
• Leigh & Williams (1964)
• 6mm wide strip of fluffless blotting or filter paper is
bent into L shape with 12 mm long foot (area 12 x 6
mm) & sterlilized.
• Dip end & foot into urine, press on to the surface of
agar medium.
• Incubate and afterwards count the colonies, 105
bacteria/ml corressponds to 25 bacilli or 30 cocci
colonies

20. Dip-Slide Method
• Dip-slide is a small plastic tray with agar medium,
opposite sides may carry different mediums.
• Mid stream urine is collected and dip-slide is
immersed in urine.
• It is sent to lab and incubated and viable counts are
estimated by representative charts.
• Convenient for peripheral labs.
• Does not provide cellular contents

21. Interpretation of urine culture
Urine
specimen &
patient
Count LE
Not likely to be
significant
Additional data
suggesting that
isolate is
significant
Midstream,
female with
cystitis
>102 CFU +
Potential pathogen
< contaminant
-
Midstream,
female with
pyelonephritis
>105 CFU +
Potential pathogen
< contaminant
Gram stain
demonstrates
potential pathogen in
neutrophils and/or
casts
Midstream,
asymptomatic
bacteriuria
>105 CFU -
<105 CFU of
potential pathogen;
Confirm by repeating
urine culture when
clinically indicated

22. Interpretation of urine culture
Urine
specimen &
patient
Count LE
Not likely to be
significant
Additional data
suggesting that isolate
is significant
Midstream,
male with UTI
>103CFU +
<103CFU of
potential
pathogen;
Gram stain demonstrates
potential pathogen in
neutrophils and/or casts
Straight
catheter, all
patients
>102CFU for
symptomatic
patients
+
<102CFU
potential
pathogen/ml,
urine LE is
negative
Gram stain demonstrates
potential pathogen in
neutrophils and/or casts
Indwelling
catheter, all
patients
>103CFU +/-
Bacteriuria
detected in
asymptomatic
patients
No reason to culture
unless patient is
symptomatic

23. Significant Bacteriuria importance
• Significant bacteriuria is applicable for only
Enterobacteriaceae group.
• Multiple (three or more) species of gram negative
bacteria- contamination in such case significant
bacteriuria is not considered.

24. Asymptomatic Bacteriuria importance
• The Infectious Diseases Society of America (IDSA) established
guidelines for the screening and treatment of asymptomatic
bacteriuria (Nicolle et. al., 2005).
• The optimal management depends significantly on specific patient
characteristics, co-morbidities, and risk factors.
• Asymptomatic bacteriuria was consistently harmful in all
populations and necessitated antibiotic treatment.
• Antibiotic treatment of asymptomatic bacteriuria in pregnant women
resulted in a significantly reduced incidence of pyelonephritis.

25. Conclusion
• Culture done- Nitrite test or LE test positive (Reflexive urine
test).
• The absence of bacteria on Gram's stain of spun urine
sediment has a high negative predictive value for significant
bacteriuria.
• Urine cultures add enormous specificity to the diagnosis of
UTI and continue to be the gold standard for diagnosis.

26. Conclusion
• A positive microscopic examination- 2 microorganisms
uniformly distributed per oil immersion field, after
observation of at least 20 fields, according to the criteria
of Washington et. al.
• Multiple (three or more) species of gram negative
bacteria- contaminated.

27. THANK YOU