Microbiology

2. SCOPE
URINE SPECIMEN COLLECTION, TYPES, METHODS,
INSTRUCTIONS AND STORAGE
URINALYSIS PROCEDURE AND INTERPRETATION
PRE-ANALYTICAL, ANALYTICAL AND POST ANALYTICAL FACTORS
IN URINALYSIS

3. INTRODUCTION
• Urinalysis is a test done to examine the physical, chemical and microscopic content of the
urine. In this context, we’ll relate it to urine R/E.
• Examination of urine is the oldest among tests in the history medical laboratory and it is used
as an aid in diagnosis and monitoring of a number of disorders or conditions that are:
a) Haematological
b) Metabolic
c) Hepatic and renal
d) Urological

4. RENAL ANATOMY AND PHYSIOLOGY
• The renal system is made up of two kidneys, two ureters, one bladder and one urethra.
• The kidneys are two bean shaped organs located under the lowermost part of the of the ribs
in the posterior abdominal cavity.
• Nephron is the function unit of the kidneys and each kidneys contains appx one million
nephrons. Each nephron consists of a glomerulus, essentially, the filtering system.
• The kidney is a highly discriminating organ, which maintains the internal environment by
selectively excreting or retaining various substance according to the specific body needs.
• Approximately 1,200 millilitres of blood flow through the kidneys each minute.

5. RENAL ANATOMY AND PHYSIOLOGY
The kidneys perform the following functions:
 Regulation of water and electrolyte (chloride, potassium, calcium, hydrogen etc)
balance as well as the regulation of acid-base balance of the blood.
 Regulation of body fluid osmolality and electrolytes
 Excretion of metabolic waste products and foreign chemical. The kidneys are
the primary means of eliminating metabolic waste products from the body.
These include urea from metabolism of amino acids, bilirubin from haemoglobin
breakdown, uric acid from nucleic acids and creatinine from muscle creatine).

6. RENAL ANATOMY AND PHYSIOLOGY
Secretion of hormones such as renin.
Gluconeogenesis, thus, synthesis of glucose of from amino
acids and other precursors including lactate and glycerol
during prolonged fasting.

7. MECHANISM OF URINE FORMATION
THREE MAIN PROCESSES
• 1. GLOMERULAR ULTRA FILTRATION
• 2. TUBULAR REABSORPTION
• 3. TUBULAR SECRETION

8. THE COMPOSITION OF URINE
• NORMAL URINE CONSTITUENTS
Water (about 95% of urine)
Urea
Creatinine
Uric acid
Electrolytes

9. THE COMPOSITION OF URINE
• ABNORMAL URINE CONSTITUENTS
Glucose
Protein
Bile pigments
Blood cells
Cast, parasites and bacterial microbes

10. FACTORS AFFECTING THE COMPOSITION OF URINE
•Diet and nutritional status
•Condition of body metabolism
•Ability of kidney function
•Level of contamination with pathogenic microorganisms or non-
pathogenic microflora

11. RENAL THRESHOLD
•This is the highest concentration of a substance, which is
present in the blood before it is found in the urine.
•Glucose has a high threshold, because it is completely
absorbed from the glomerular filtrate and only found in urine
when blood glucose level is markedly raised.

12. The quality of a sample is as good as the results. For reliable urinalysis, the urine must be
properly collected.
• A freshly voided urine specimen is adequate for most urinalysis.
Generally, about 10mls of urine is required for manual routine urinalysis.
• Catheterised urine collection
• Suprapubic transabdominal needle aspiration
• Paediatric collection, with special polyethlene bags for collection
• 24-hour urine collection.
URINE COLLECTION METHODS

13. TYPES OF URINE SPECIMEN
• FIRST MORNING SPECIMEN – obtained during the first urination of the day. Most
concentrated, bladder incubated. Best for nitrite, protein and microscopic examination.
• RANDOM SPECIMEN – obtained at any time during the examination. Most convenient,
most common. Best for chemical screen and microscopic examination.
• DOUBLE/SECOND-VOIDED URINE – in this case, first morning specimen discarded and
the second specimen collected and test. Best for reflection of blood glucose and keeping
of formed elements intact.

14. TYPES OF URINE SPECIMEN
• POSTPRANDIAL – a specimen obtained 2 hours after meal. Good for glucose
estimation.
• 24 HOUR SPECIMEN – obtained within 24 hours. Necessary for quantitative test,
especially for quantitative determination of protein.
• MID-STREAM SPECIMEN – obtained from the middle part of the first urine. Best for
routine urinalysis, also important for bacteriological urine culture.

15. TYPES OF URINE SPECIMEN
CLEAN CATCH SPECIMEN – this is the best method used for microbial culture and routine
urinalysis. This method is collected as follows:
• The genital area should be cleaned with soap and water and rinsed well in other to keep off
bacteria on the skin from contaminating the specimen.
• The patient should urinate a small amount and this is discarded.
• The urine that comes next, the mid-stream, should be collected into a sterile container of 30 to
50mls
• After collecting the needed amount, the patient continues to urinate and this discarded.

16. URINE COLLECTION INSTRUCTIONS
Garbage in, garbage out therefore the instructions are very vital.
There will be different instructions for the same test based on:
• Medical history
• Diagnosis (presumptive or confirmed)
• Age
• Medical condition.
• Record time of urine collection and receipt at lab

17. SOURCES OF ERROR IN THE COLLECTION OF URINE
• Bacteriologically or chemically contaminated specimen
• Wrong type/amount of preservative
• Partial loss of specimen or inclusion of two-morning specimen in the 24-hour collection
• Inadequate mixing of specimen before examination
• Careless measuring of the 24-hour volume.

18. URINE STORAGE AND VIABILITY
Long standing of urine at room temperature can cause:
• Growth of bacteria
• Break down of urea to ammonia by bacteria leading to an increase in the ph of the urine
and this may cause the precipitation of calcium and phosphates.
• Oxidation of urobilingen to urobilin.
• Usage of glucose by cellular elements
• Lysis of RBCs, WBCs and casts

19. METHODS OF PRESEVATION OF URINE SPECIMEN
PHYSICAL METHOD
• Refrigeration
• Freezing
CHEMICAL METHOD
Use of chemical preservatives such as:
• Thymol
• Toluene
• Formaldehyde
• Hydrochloric acid
• Chloroform
• Boric acid
• Chlorhexidine
• Sodium carbonate

21. URINALYSIS
Urinalysis requires 2 broad types of examination:
1. MACROSCOPY
• Direct visual observation to note colour, appearance, odor and consistency.
• Dipstick analysis: tests include pH, specific gravity, protein, glucose, ketones, nitrite, and
leukocyte esterase etc.
2. MICROSCOPIC ANALYSIS
• Sediment is examined for red blood cells, white blood cells, epithelial cells, casts, bacteria,
yeast, and crystals, and others using the microscope.

22. PROCEDURE
• Gather and check expiry dates of reagents or strips, perform hand hygiene and put on
PPE’s
• Confirm patients labelled information; name, age, dob, path number etc
• Inspect colour by picking the specimen container up and bring it to the eye level as much
as possible.
• Inspect clarity and assess urine odour
• Gently swirl specimen container
• Remove test strip and immerse into urine ensuring that all zones are immersed.

23. PROCEDURE CONT’D
• Remove test strip and ensure that excess urine is removed.
• Place strip on a horizontal orientation to avoid cross contamination of zones.
• Inspect each test at the appropriate time interval using the dipstick analysis chart.
• Discard strip, perform hand hygiene and document the results

24. Dipstick analysis

25. MACROSCOPY – COLOUR AND APPEARANCE
• Normal urine colour is often described as straw, yellow or amber. Urochrome pigment is
mainly responsible for normal urine colour. This colour may be altered by medications, food
sources or disease.
• Vitamin tablets often result in a bright yellow urine, as does the presence of bilirubin (a bile
pigment). Blood stained urine may be due to blood, haemoglobin, or beetroot. Iron
supplements may cause a dark brown specimen
• Normal urine is also transparent/clear. Turbid or cloudy urine may result from infection, the
presence of blood cells, bacteria or yeast (eg candida) etc
• Other common causes of urine turbidity: a. Semen, or vaginal discharge mixed with urine.
Urine specimen, standing for long period on the bench, will become hazy or cloudy due to
precipitation of crystals.
• MACROSCOPY IS CLOSELY RELATED TO MICROSCOPY.

26. MACROSCOPY – pH
• It is important that a fresh sample be used as urine becomes more alkaline over time as
bacteria convert urea to ammonia (which is very alkaline).
• Low pH (acidic urine) are caused by foods such as acidic fruits and other causes include
diabetes and starvation.
• High pH (alkaline) can be associated with low carbohydrate or vegetarian diet, renal
calculi. Or urinary tract infection

27. MACROSCOPY – SPECIFIC GRAVITY
• This measures the kidney's ability to concentrate or dilute urine in relation to plasma by
comparing the weight of urine (particles) to the weight of distilled water (1.000).
• Usually, 1.015 with normal fluid intake.
• Increased SG may be due to dehydration or an increased secretion of anti-diuretic
hormone (ADH)
• Decreased SG may be due to; excessive fluid intake (oral or iv fluids), renal failure, acute
glomerulonephritis, pyelonephritis, acute tubular necrosis, diabetes insipidus

28. MACROSCOPY – PROTEIN
• Normally, urine is free of protein or has only a trace.
• Proteinuria occurs with renal disease.
• While both albumin and globulin may be excreted in the urine, albumin filters more
readily than globulin, so protein in the urine is primarily albumin.
• The first indication of renal disease is often the finding of small amounts of albumin in the
urine (microalbuminuria)

29. MACROSCOPY – KETONES
They are produced during fat metabolism.
Ketones may be present in the following conditions:
• Diabetes
• Alcoholism
• A state of starvation and pregnancy.

30. MACROSCOPY – BILIRUBIN
• Presence of bilirubin in the urine may indicate
- Liver disease,
- Biliary tract infection
- Pancreatic causes of obstructive jaundice.
• Note that urine containing bilirubin will typically be red-brown in color and produce a yellow
foam when shaken.
• Bilirubin is not stable in solution, and when there is a delay in the analysis it will be oxidized
to biliverdin, which is a green pigment when exposed to light

31. MACROSCOPY – UROBILINOGEN
• Normally 1-4 mg of urobilinogen is excreted in the urine each day.
• Urine is often tested for increasing urobilinogen when investigating haemolytic jaundice
or liver disorder in which liver function is impaired.

32. MACROSCOPY – BLOOD/HAEMOGLOBIN
• Hemoglobin appears in the urine when there is extensive or rapid destruction
(hemolysis) of circulating erythrocytes that the reticuloendothelial system cannot
metabolize or store the excessive amounts of free haemoglobin.
• Hemoglobinuria is usually related to hematuria- a condition when intact red blood cells
are present in the urine.
• Red cells rapidly lysis in urine, especially when it has a specific gravity of 1.006 or less or
is alkaline.
• It may be present as a result of hemolysis in the blood stream, in a particular organ, in
the kidney of lower urinary tract.

33. MACROSCOPY – LEUCOCYTES
• Detects white cells in the urine (pyuria) which is associated with urinary tract infection.
• False positive may occur if the dipstick has been exposed to air for a long period, the
specimen is contaminated with vaginal secretions, or the patient is taking
phenazopyridine.
• False negatives can occur with increased specific gravity, decreased ph (<6.0),
increased urobilinogen, and vitamin c

34. MACROSCOPY – NITRITE
• The detection of nitrite in the urine can be used to indicate the presence of bacteria such
as escherchia coli, proteius, klebsiella, enterobacter, citrobacter, and salmonella will
reduce urinary nitrate to nitrite
• Detection rate of nitrites increases when the urine is held in the bladder for at least 4-6
hours, test for nitrite may therefore be performed on a first morning urine specimen if it is
strongly suspected but it tests negative in a random sample

35. MACROSCOPY – GLUCOSE
• Glucose is not normally present in the urine.
• Once the level of glucose in the blood reaches a r̃enal threshold, the kidneys begin to
excrete it into the urine in an attempt to decrease the blood concentration.
• Glucosuria is associated with conditions like: diabetes, liver disease, hyperthyroidism,
medications such as tetracycline, lithium, penicillin, cephalosporins, pregnancy

36. MICROSCOPY
• A microscopic examination of urine sediment detects the presence and amounts of: cells,
microorganisms, spermatozoa, mucus, casts, crystals.
• Particles of nephrological importance in microscopy
- cells
- lipids
- casts
- crystals
- microorganisms

37. THANK YOU