Culture of Urine Specimens

Exam of a Gram-stained smear

Put together and look at a Gram-stained smear of the urine while microorganism and, or white cells are visible in the wet preparation.

• switch a drop of the urine sediment to a slide and spread it to make a thin smear. permit to air dry blanketed from insects and dirt. warmth restore or methanol repair the smear and stain it by using the Gram method.
• observe the smear first with the 40_ objective to look the distribution of cloth, and then with the oil immersion objective. look specifically for bacteria related to urinary infections especially Gram-negative rods. once in a while Gram tremendous cocci and streptococci may be seen.

Notice: Usually simplest an unknown sort of organism is present in simple acute urinary infections. a couple of sort of organism is frequently seen in chronic and recurring infections. Vaginal contamination of the specimen is indicated through a mixed bacterial plant life (including Gram nice rods) and frequently the presence of epithelial cells. Neisseria gonorrhoeae in urine In male sufferers with acute urethritis, it's far regularly possible to make a presumptive diagnosis of gonorrhoea by way of locating Gram-negative intracellular diplococci in pus cells handed in urine .

Take a look at the specimen biochemically

Biochemical assessments which might be useful in investigating UTI include:

• Protein
• Nitrite
• Leukocyte esterase Protein

Protein

Most laboratories check urine mechanically for protein the use of sulphosalicylic acid reagent or a protein reagent strip test. Proteinuria is located in most bacterial urinary tract infections. Different causes include glomerulonephritis, nephrotic syndrome, eclampsia, urinary schistosomiasis, high blood pressure, extreme febrile illnesses, HIV-related renal sickness and treatment with nephrotoxic antiretroviral drugs.

Nitrite

Urinary pathogens, e.g. E. coli (commonest reason of UTI), Proteus species, and Klebsiella species, are capable of reducing the nitrate normally found in urine to nitrite. This will be detected by the Greiss test or a nitrite reagent strip test,imparting the organisms are present in the urine in enough attention. Whilst first morning urine is examined, approximately 80–90% of UTI because of nitrate-lowering pathogens can be detected. The check is bad whilst the infection is due to pathogens that do not reduce nitrate which includes Enterococcus faecalis, Pseudomonas species, Staphylococcus species and Candida organisms, or while as previously referred to the bacteria are too few inside the urine. now and again the nitrite check is terrible because nitrate is missing inside the urine due Leukocyte esterase (LE) This enzyme is unique for polymorphonuclear neutrophils (pus cells). It detects the enzyme from both active and lyzed WBCs. LE testing is an alternative approach to detecting pyuria when it is not viable to look at sparkling urine microscopically for white cells or when the urine isn't clean and probably to contain normally lyzed WBCs. LE may be detected the usage of a reagent strip test consisting of the Combur 2 check LN (Roche strip) which detects both nitrite and leukocytes (LE) or a multitest reagent strip with an area for leukocyte . Fake negative strip test effects can arise when the urine carries boric acid or excessive amounts of protein (_500 mg/one hundred ml) or glucose (_2 g/100 ml)

Culture the specimen

It isn't always essential to culture urine which is microscopically and biochemically normal, except while screening for asymptomatic bacteriuria in pregnancy. Culture is required while the urine incorporates bacteria (as indicated by means of the Gram smear), cells, casts, protein, nitrite, or has a markedly alkaline or acid reaction.

Estimating bacterial numbers

It's far necessary to estimate the approximate wide variety of microorganism in urine because normal specimens may also comprise small numbers of contaminating organisms, generally much less than 10 000 (104) consistent with ml of urine. Urine from someone with an untreated acute urinary infection usually contains one hundred 000 (one hundred and five) or more microorganism in keeping with ml. The approximate quantity of bacteria in keeping with ml of urine may be envisioned by using the use of a calibrated loop or a measured piece of filter out a paper. Both strategies are based on accepting that a unstained colony represents one organism. as an example, if an inoculum of ml produces 20 colonies, the quantity of organisms represented in ml of urine is 20, or 10 000 in 1 ml (500 _ 20). The calibrated loop approach the use of quarter plates of the way of life media is usually recommended due to the fact it's far inexpensive, easy to carry out, and presents person colonies which can be less complicated to identify and remove for antimicrobial susceptibility test.

Cystine lactose electrolyte-deficient (CLED) agar

• mix the urine (freshly accrued clean-seize specimen) by rotating the container.
• using a sterile calibrated twine loop, e.g. one which holds ml (zero.002 ml), inoculate a loopful of urine on a quarter plate of CLED agar . If microscopy suggests many microorganism, use a half plate of a medium.
• Incubate the plate aerobically at 35–37 °C in a single day.

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Day 2 and Onwards

Have a look at and document the cultures CLED agar subculture appearance in particular for colonies that would be:

• Escherichia coli (perform indole and beta-glucaronidase tests for speedy identification)
• Proteus species.
• Pseudomonas aeruginosa.
• Klebsiella lines.
• Staphylococcus aureus, .
• Staphylococcus saprophyticus.
• Enterococcus faecalis.

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Note: Contaminating organisms usually produce some colonies of mixed increase. most urinary infections show the culture of a single sort of organism despite the fact that combined infections can arise mainly in continual infections or follow catheterization or gynaecological surgical treatment.

Reporting bacterial numbers

Remember the approximate range of colonies. Estimate the wide variety of microorganism, i.e. colony-forming units (CFU) consistent with ml of urine. document the bacterial count number as:

• much less than 10 000 organisms/ml (104/ml), now not enormous.
• 10 000–100 000/ml (104–105/ml), dubious importance (endorse repeat specimen)
• greater than 100 000/ml (one zero five/ml), giant bacteriuria.

Example

If 25 E. coli colonies are counted and a ml loop become used, the approximate range of CFU in step with ml of urine: 500 _ 25 _ 12 500 this sort of rely upon could be reported as: 10 000–100 000 E. coli/ml

Antimicrobial susceptibility test

Carry out susceptibility testing on urines with widespread bacteriuria, mainly from patients with a history of habitual UTI. Cultures from sufferers with a number one uncomplicated UTI may not require a susceptibility take a look at.

Possible pathogens

BACTERIA

Gram positive

• Staphylococcus
• Saprophyticus
• Haemolytic streptococci

Gram negative

• Escherichia coli
• Proteus species
• Pseudomonas aeruginosa
• Klebsiella strains
• Salmonella Typhi
• Salmonella Paratyphi
• Neisseria gonorrhoeae

References:

D greenwood, Slack RCB and J Peutherer.Medical microbiology.2001.

JG College, AG Fraser and BP Marmion.Practical Medical microbiology.Fourteenth Edition. Churchill Livingstone, 1996.

JP Micheal, ECS Chan and NR Krieg.Microbiology.Fifth Edition. Delhi: Mcgraw-hill, 1993.

M Cheesbrugh.Medical laboratory manual for tropical countries.London, 2007.