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MLW.SOP.L.MICRO.

016 Version 1 Page 2 of 10


Contents

1. BACKGROUND .................................................................................. 3
2. PURPOSE ........................................................................................... 4
3. RESPONSIBILITIES ........................................................................... 4
4. PROCEDURE ..................................................................................... 4
4.1 MATERIALS NEEDED ................................................................... 4
4.2 STORAGE CONDITIONS .............................................................. 4
4.3 TEST PROCEDURE ...................................................................... 4
4.4 QUALITY CONTROL ...................................................................... 5
4.5 INTERPRETATION OF RESULTS ................................................. 5
4.6 LIMITATIONS ................................................................................. 6
5. HEALTH & SAFETY ........................................................................... 6
6. ASSOCIATED PROCEDURES .......................................................... 6
7. REFERENCES .................................................................................... 7
8. APPENDICES ..................................................................................... 8
Appendix 1: Fastread 102 counting chamber instructions for use .... 8
9. Training Record for this SOP ......................................................... 10

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1. BACKGROUND
Urinary tract infection (UTI) results from the presence and multiplication of
microorganisms in one or more structures of the urinary tract with associated tissue
invasion. This can give rise to a wide variety of clinical syndromes. These include
acute and chronic pyelonephritis (kidney and renal pelvis), cystitis (bladder),
urethritis (urethra), epididymitis (epididymis) and prostatitis (prostate gland). Infection
may spread to surrounding tissues (e.g. perinephric abscess) or to the bloodstream.

Protection against infection is normally given by the constant flow of urine and
regular bladder emptying. Urine is a poor culture medium for many bacteria due to
its acidity, high urea concentration and variable osmolality and, in men, possibly
partly as a result of antibacterial activity of prostatic secretions
1
.

Bacteriuria Implies that bacteria are present and may be cultured from urine. The
patient may or may not be symptomatic.
Symptomatic patients They may be bacteriuric or abacteriuric. Symptoms in
children and the elderly, when present, may be non-specific and difficult to interpret.
Frequency The average bladder capacity is about 500 mL. Significant reduction
in capacity accompanies acute inflammation which can lead to an increase in the
frequency of micturition.
Dysuria Painful and difficult micturition.
Urgency A strong desire to empty the bladder, which can lead to incontinence.
Nocturia Waking in the night one or more times to void the bladder
2
.
Nocturnal enuresis The involuntary voiding of urine during sleep, i.e. bed-wetting.
Incontinence The involuntary leakage of urine. The commonest form of this is
stress incontinence where leakage accompanies an increase in intra-abdominal
pressure due to sneezing, coughing or laughing. Overflow or dribbling incontinence
accompanies an overfilled bladder.
Prostatism Symptoms include: hesitancy or delay in initiating micturition,
intermittency or interruption, and reduced force of the urine stream resulting from
prostate gland pathology.
Renal colic This is characterised by very severe cramping pain resulting from
distension of the ureter and pelvis above an obstruction such as a renal stone.
Often accompanied by frequency and urgency.

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Asymptomatic bacteriuria Common in several patient groups, particularly the
elderly, pregnant women and diabetic patients.


2. PURPOSE
This SOP explains how to perform Urine microscopy and culture on mid-stream
urine samples received at the MLW laboratory. If any other type of urine specimen
is received this should only be processed after discussion with, and advice
from, the clinical microbiologist.


3. RESPONSIBILITIES
1) Laboratory technicians
2) Visiting clinical scientists


4. PROCEDURE
4.1 MATERIALS NEEDED
1) Fastread 102 counting chamber
2) Blood agar plate
3) CLED agar plate
4) Disposable 1l sterile loop
5) Pipette


4.2 STORAGE CONDITIONS
1) Agar plates should be stored at 2-8oC and used within 1 month of their
production.
2) All other materials are stored at room temperature


4.3 TEST PROCEDURE

Day one:
1) Gently mix the urine sample by inverting the tube several times (do not
shake the sample)

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2) Perform a cell count using a Fastread 102 counting chamber (see
appendix 1)
3) Place 1 drop of urine using a pipette into the sample application area of
the chosen counting well and allow it to distribute evenly throughout the
counting area.
4) Count the numbers of cells (white cells and red cells) across all ten large
grids (4x4 small grids) to achieve a count per l or count a smaller area
and do the appropriate calculation to achieve a count per l.
5) To achieve a count per ml multiply the count per l by 1000.
6) Inoculate 1l of urine sample onto a blood agar plate and a CLED agar
plate using a 1l sterile loop and spread to achieve single colony growth
7) Incubate both plates in air at 37
o
C overnight

Day two:
1) Check both plates for bacterial growth and identify the organisms
according to interpretation of results (4.5) below.
2) Perform sensitivity tests on significant bacterial growth (as defined by the
table in interpretation of results below) using MLW.SOP.MICRO.208.

Day three:
1) Read and interpret sensitivity tests as describe in MLW.SOP.MICRO.208


4.4 QUALITY CONTROL
See associated procedures


4.5 INTERPRETATION OF RESULTS
Identification of bacterial growth and subsequent sensitivity testing should only be
done if the bacterial growth is considered significant in conjunction with the
microscopic analysis (see table below).

White cell count Culture result (per agar plate) Significant

Any Pure single organism 10 colonies Yes

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Any Pure single organism <10 colonies No
10
5
per ml 2 types of organisms 10 colonies Yes
10
5
per ml More than 2 types of organisms 10 colonies No
<10
5
per ml Mixed culture 10 colonies No
Any Mixed culture < 10 colonies No

Reporting:
Bacterial Growth Report as:

GNB, Yellow on CLED Lactose fermenting coliform
GNB, Clear on CLED Identify (MLW.SOP.MICRO.204)
GPC Identify (MLW.SOP.MICRO.203)
Mixed culture (not significant) No significant growth
No growth No bacterial growth


4.6 LIMITATIONS
1) Mid-stream urines give more reliable microscopy results
2) The urine must be tested within 24 hours of collection as contaminating
bacterial overgrowth may obscure significant organisms after this time
unless collected into a sample tube containing boric acid preservative.
3) Urine samples recovered from nappies are NOT suitable for
examination


5. HEALTH & SAFETY
1) Follow health and safety guidelines as laid down in the MLW Laboratory
Health and Safety Manual


6. ASSOCIATED PROCEDURES
1) MLW.SOP.MICRO.203 (Isolate identification - Gram positive cocci)
2) MLW.SOP.MICRO.204 (Isolate identification - Gram negative bacilli)


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7. REFERENCES

1. Sobel JD. Pathogenesis of urinary tract infections. Host defenses. Infect Dis
Clin North Am 1987;1:751-72.
2. Van Kerrebroeck P, Abrams P, Chaikin D, Donovan J, Fonda D, Jackson S,
et al. Nocturia. Neurourology Urodynamics 2002;21:179-83.

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8. APPENDICES
Appendix 1: Fastread 102 counting chamber instructions for use



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9. TRAINING RECORD FOR THIS SOP

The following have been trained in this SOP and have demonstrated adequate
knowledge of its contents:

Name of Trainee Name of Trainer Signature/Date of
Trainee
Signature/Date of
Trainer*