Once you have submitted your urine sample for analysis, it will be sent off to the local laboratory or a hospital laboratory if you are an in-patient or outpatient.
The standard length of time for a laboratory culture analysis is around 18-24 hours. Your sample may not immediately be sent to the laboratory but rely on a once-daily collection from the GP surgery.
How your urine is analysed in the laboratory
When it arrives, your sample will be analysed by its appearance (colour, cloudiness, smell), another dipstick check carried out and finally, macroscopic analysis.
Normal urine is usually a light yellow in colour and clear without any cloudiness. Any change in this can show:
- a possible infection (cloudy urine)
- dehydration (dark urine colour)
- red blood in the urine, also referred to as hemaeturia (for women this can be due to menstrual blood)
- liver disease (urine can look the colour of tea)
- breakdown of muscle (orange or tea coloured urine)
- Certain medications, foods and vitamins (Vitamin D) can also change the colour. Very foamy urine may represent large amounts of protein in the urine (proteinuria)
Another dipstick is applied to the urine (as your GP will have done in the surgery) to check for signs of infection or inflammation (white and/or red blood cells, protein and nitrates) including the pH of the urine. Additionally, the dipstick reagent pads test for the presence of Bilirubin, Urobilinogen, Ketones and Glucose.
A neutral pH is 7.0. The higher the number, the more alkaline it is. The lower the number, the more acidic your urine is. The average urine sample tests at about 5.0 as the urine is slightly acidic. This is due to the normal daily acid production in the body to maintain an acid-base balance. Therefore, any abnormalities in the acid-base balance has a direct effect on urinary pH levels.
These levels are particularly useful in the evaluation of stones, crystals or infection. For example, in a patient with a possible kidney stone, the urinary pH level is helpful. The main types of kidney stones are:
- calcium stones, the most common type of stone
- struvite stones, usually caused by an infection, like a urine infection
- uric acid stones, usually caused by a large amount of acid in your urine
Uric acid, cystine, and calcium oxalate stones tend to form in acidic urine, whereas struvite (magnesium ammonium phosphate) and calcium phosphate stones form in alkaline urine.
People with a UTI due to Proteus and Klebsiella bacteria typically have alkaline urine whereas bacteria such as E-coli are usually found in a more acidic environment.
This pH will also be noted on the report. However, pH is also affected by diet; a high protein intake can give rise to acidic urine, whereas a high intake of dairy products or vegetables can give rise to alkaline urine.
White Blood Cells (Leukocytes)
Only a few white blood cells are normally present in urine. When these numbers increase, the dipstick test will become positive. This indicates that there is inflammation in the urinary tract or kidneys and the body is excreting more white blood cells. In addition to possible infection, white blood cells/leukocytes can also indicate chronic kidney inflammation caused by a kidney stone, a tumour of the kidneys, bladder or urethra, infections such as chlamydia or other sexually transmitted diseases and fungal infections such as Thrush.
Protein in the urine may be a sign of kidney disease. The protein test pad provides a rough estimate of the amount of albumin in the urine. Albumin makes up about 60% of the total protein in the blood. Normally, there will be no protein or a small amount of protein in the urine. When urine protein is elevated, a person has a condition called proteinuria.
Small amounts of albumin may be found in the urine when kidney dysfunction begins to develop. If it is felt necessary the laboratory may request that a further, more detailed urine albumin test be carried out by the GP or hospital. The urine albumin test is more sensitive than a dipstick urinalysis and is routinely used to screen people with chronic conditions that put them at risk for kidney disease, such as diabetes and high blood pressure. Protein in the urine can also be due to dehydration, pregnancy, disease of the heart and some cancers.
Urine will contain a certain amount of nitrate usually down to foodstuffs but, in a normal urine sample, this type of nitrite will be indicated as ‘absent’ or ‘not present’ on a dipstick. But, in certain instances, nitrates can be indicated as being ‘present’ or “+”. The most common occurrence of positive nitrites in urine is in the presence of bacteria which convert the non-ionic nitrate into nitrite.
Bacteria where nitrates would be shown as positive on dipstick include certain species of E. coli, Klebsiella, Proteus or Pseudomonas.
However not all bacteria convert nitrates in the urine so relying on this marker in the testing process does not exclude infection.
An important point to note is the conversion of nitrates in the urine can take up to four hours and a fresh sample of urine may not readily show this if the sample is immediately analysed by the laboratory.
Bilirubin and urobilinogen
Bilirubin is a chemical produced when red blood cells are broken down. It is transported in the blood to the liver, where it is processed and excreted into the gut as a constituent of bile. In the gut, bacteria act on the bilirubin to transform it into urobilinogen. It is usual for urine to contain urobilinogen but not bilirubin. Bilirubin in the urine may be an indicator of a breakdown of red blood cells. It may not be effectively removed by the liver, which may suggest liver disease or a problem with drainage of bile into the gut, such as gall stones.
These are chemicals that are formed during the abnormal breakdown of fat and are not normal constituents of urine.
Breakdown of fat may result from prolonged vomiting, fasting or starvation; individuals on a diet or who present with diarrhoea and vomiting may have a positive result.
Ketones can also be present in the urine of people with poorly controlled diabetes. This can make the blood more acidic and is known as diabetic ketoacidosis; it should be reviewed urgently by a doctor. Some medications, such as captopril, may also produce a false positive result (Steggall, 2007).
Glucose in the urine (glycosuria) can occur in pregnancy or patients taking corticosteroids. It may also be indicative of diabetes but is not normally found in a urine sample. Although glycosuria is an indication of endocrine abnormality, it is not diagnostic and further investigation, such as fasting blood tests, may be required.
Once this dipstick check has been carried out, a microscopic examination is the next step using urine sediment. To do this, the urine sample is centrifuged (spun) to concentrate the substances in it at the bottom of a tube. The fluid at the top of the tube is then disposed of and the drops of fluid remaining are examined under a microscope and the following will be noted on your urine sample report:
Red blood cells (RBCs)
Normally, a few RBCs are present in urine. Inflammation, injury, or disease in the kidneys or elsewhere in the urinary tract, for example in the bladder or urethra, can cause RBCs to leak out of the blood vessels into the urine. However, RBCs can also be due to blood from haemorrhoids or menstruation, kidney, bladder or urethral cancers, enlargement of the prostrate in men; kidney stones and certain blood diseases such as sickle cell anaemia.
White blood cells (WBCs or leukocytes)
The number of WBCs in urine is normally low. When the number is high, it indicates an infection or inflammation somewhere in the urinary tract due to the immune system response.The agreed total number of leukocytes to be present in a millilitre of urine to establish the baseline for infection remains unchanged since the work of C Dukes in 1928. He proposed a threshold of less than 10 wbc/per millilitre of urine as the upper limit of normal white blood cell excretion in the urine. Above that limit and the sample may be positive for infection.
Also note that White Blood Cell or Leukocytes can also indicate chronic kidney inflammation caused by a kidney stone, a tumour of the kidneys, bladder or urethra, infections such as chlamydia or other sexually transmitted diseases and fungal infections such as Thrush.
Epithelial cells can be either squamous, originating from the vagina, urethra or genital skin, urothelial, that is cells from the bladder wall and finally renal tubular originating from the kidneys.When analysing the sample, significant epithelial cells may lead to further analysis to identify their type. Squamous epithelial cells originate from the urethra or vagina and are commonly found in urine samples due to transfer whilst providing the sample – these are often reported as a contaminate in the sample without specific analysis of the actual type of epithelial cell. However, it’s the presence of urothelial epithelial cells that would indicate a urinary tract infection. Or, if many renal (kidney) epithelial cells are discovered in urinalysis, then it could indicate a viral infection or problem with the kidneys.
Casts are particles that are formed from protein secreted by kidney cells. Under the microscope, they often look like a sausage shape because of the way they form and in healthy people they appear nearly clear. This type of cast is called a ‘hyaline’ cast. When an infection is present in the kidney, other things such as RBCs or WBCs can become trapped in the protein as the cast is formed. When this happens, the cast is identified by the substances inside it, for example as a red blood cell cast or white blood cell cast. Different types of casts are associated with different kidney diseases and the type of casts found in the urine may give clues as to which is affecting the kidney. Normally, healthy people may have a few casts. After strenuous exercise, more casts may be detected. Cellular casts, such as RBC and WBC casts, indicate a kidney disorder.
Urine contains many dissolved substances (solutes) – waste chemicals that your body needs to eliminate after filtration through the kidneys.These can form crystals and they may group together to form kidney “stones”. These stones can become lodged in the kidney itself or in the ureters – tubes that pass the urine from kidney to the bladder – causing extreme pain. Medications, drugs, and x-ray dye can also crystallize in urine.
Bacterial incubation on petri dish
If the microscopic examination shows signs of infection then a sample of the urine sediment will be placed on a Petri dish and left to incubate over 18 -24 hours. Bacteria can reproduce very quickly given the right conditions, such as warmth, moisture and suitable nutrients.
To ensure the cultures are not contaminated by other microorganisms, the following sterile conditions are needed; the Petri dishes, nutrient agar jelly inside these dishes and other culture media must be sterilised.
The inoculating loops used to transfer microorganisms to the petri dish must be sterilised (usually by passing the metal loop through a Bunsen burner flame) and finally, the lid of the Petri dish is sealed with sticky tape to stop microorganisms from the air getting in and contaminating the culture.
Bacteria will produce colonies differing in appearance as they grow, some colonies may be coloured, some colonies are circular in shape, and others are irregular. Different bacterial strains produce these characteristics. The sample from the petri dish is then “gram stained” to identify the actual strain of bacteria. This means a crystal violet colour stain is applied to the sample and placed under a microscope.
All bacteria are described as either Gram-negative or Gram-positive
Gram-positive bacteria remain purple. Gram-negative bacteria are stained pink. Gram-negative bacterial strains include:
- Escherichia coli
- Klebsiella pneumoniae
- Proteus mirabilis
- Pseudomonas species
- Morganella morganii
- Citrobacter species
Gram-positive strains include:
- Enterococcus faecalis
- Enterococcus faecium (E. faecium)
- Staphylococcus aureus
- Staph saprophyticus
Under the microscope, the appearance of bacteria is observed. The lab can then finally determine:
- Are they Gram-positive or negative?
- What are the physical characteristics?
- Are the cells individual or are they in chains, pairs etc.?
- How many are there and how large are the cells?
The final part of the process is to then count the total colony numbers of the single bacteria identified.
These are known as colony-forming units usually abbreviated as CFU. For a positive diagnosis to be made of an infection, the agreed standard is currently greater than >10 5 /ml in a millilitre of urine. Up to 10,000 colonies of bacteria/ml are considered normal. Greater than 100,000 colonies/ml represents a positive urinary tract or kidney infection.
For counts between 10,000 and 100,000, the culture is indeterminate and results will show low growth or mixed growth.
If sufficient bacteria are grown and the single pure strain identified e.g. E-coli, antibiotics will be tested against the bacteria to check their effectiveness in stopping the infection. This is known as susceptibility and resistance and the results will be noted on the lab report to help your GP prescribe the correct antibiotic.
Yeast can also be present in urine. If yeast is found in urine, then the laboratory may recommend tests for a yeast (fungal) infection on vaginal secretions or may culture on a petri dish to identify the yeast and whether it could be causing UTI or other disease symptoms.
Antibiotic testing/bacterial susceptability
If sufficient bacteria are grown and the single pure strain identified such as e coli or enterococcus, antibiotics will be tested against the bacteria to check their effectiveness in stopping the infection. This is known as susceptibility and resistance and the results will be noted on the lab report to help your GP prescribe the correct antibiotic.
To determine bacterial susceptibility, the laboratory injects the bacteria isolated from the sample into a series of tubes or cups that contain broth dilutions of the antibiotic. After a standardized incubation period, the lowest concentration of antibiotic that prevents visible growth of the organism is classified as the minimal inhibitory concentration (MIC).
The alternate method of bacterial susceptibility is via the disk diffusion method. Using this technique, disk plates are impregnated with various antibiotics and placed on the surface of an agar plate that has been injected with the isolated bacterial sample. The antibiotic diffuses outward from the disk over a standard incubation time, and the diameter of the zone of inhibition is measured. The size of this zone is compared with standards to determine the sensitivity of the organism to the drug.
The laboratory report that will be sent to your GP will report the name of bacterium grown and the sensitivities of the antibiotics tested against each bacterium.
The interpretation of this testing categorizes each antibiotic result as susceptible (S), intermediate (I), sensitive-dose dependent (SD), resistant (R) or no interpretation (NI). What does this mean?
- Susceptible (S): This indicates that the antibiotic may be an appropriate choice for treating the infection caused by the bacteria tested. i.e. the organism is likely to respond to treatment with this drug, at the recommended dosage. Bacterial resistance is absent or at a clinically insignificant level.
- Intermediate (I): It is applicable to those is bacteria that are “moderately susceptible” to an antibiotic. The intermediate category serves as a buffer zone between susceptible and resistant. The antimicrobial agent may still be effective against the tested isolate but response rates may be lower than for susceptible isolates.
- Susceptible-dose dependent (SDD):
This is a new category for antibacterial susceptibility testing. If a particular bacteria falls under this category, the susceptibility will depend on the dosing regimen used. Higher doses or more frequent doses or both should be used to achieve concentration levels that are more likely to be clinically effective.
- Resistant (R). If a bacteria is resistant to a particular antibiotic; it won’t be inhibited by that specific medication using a normal dosage. There is also no expectation of this bacteria to respond a higher dosage.
- Non-susceptible (NS) This category is used for bacteria where there are resistant strains.
Microbiology report for GP and patient
Once the laboratory analysis has been finished the report is sent to your GP. If the report is positive for bacterial infection, you will be contacted to visit the GP and a prescription issued for the identified antibiotic from the laboratory report to treat your UTI. If the report details the possibility of kidney stones, you may be referred by the GP for further tests and treatment particularly if the stone is causing considerable pain. If the sample is negative, then if symptoms persist, you may request a retest from your GP.
A sample report: