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Culturing of Urine, Skill based Microbiology

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Culturing of urine skill based learning l.jpg
Culturing of urineskill based learning

Dr.T.V.Rao.MD

Dr.T.V.Rao MD


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Over view of Urinary Tract Infections

Most common infectious disease

Most Numerous specimens are received in the Laboratory

30 to 40 % of specimens received in Microbiology laboratories are Urine specimens, to Identify the Infection.

Diagnostic information is important for the clinician.

Appropriate clinical information gives many clues for better diagnostic evaluations.

Specimen collection is the primary objective in getting an ideal sample.

Dr.T.V.Rao MD


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What is urinary tract infection

  • an infection of one or more structures in the urinary system. Most UTIs are caused by gram-negative bacteria, most commonly Escherichia coli or species of Klebsiella, Proteus, Pseudomonas, or Enterobacter, although other strains, such as Staphlyococcus and Serratia, are emerging

Dr.T.V.Rao MD


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Urinary tract infections are more common in women

  • The condition is more common in women than in men. UTI may be asymptomatic but is usually characterized by urinary frequency, burning pain with voiding, and, if the infection is severe, visible blood and pus in the urine. Fever and back pain often accompany kidney infections

Dr.T.V.Rao MD


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Common Uropathogens

  • Escherichia coli

  • Other Enterobacteriaceae (Klebsiella, Enterobacter, Proteus, Citrobacter)

  • Pseudomonas aeruginosa

  • Enterococcus

  • Staphylococcus saprophyticus

  • Staphylococcus aureus

  • Streptococcus agalactiae (group B)

  • Candida

    Associated with staphylococcemia

    Denotes vaginal colonization in pregnant women

Dr.T.V.Rao MD


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Uncommon UropathogensM.tuberculosis produces chronic infection

  • Corynebacterium urealyticum1

  • Haemophilus influenza and H. parainfluenzae2

  • Blastomyces dermatitidis3

  • Neisseria gonorrhaeae4

  • Mycobacterium tuberculosis5

Dr.T.V.Rao MD


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Pathophysiology of urinary tract infection

  • Ascending route of infection most common

  • Colonization of urethra and periurethral tissue by uropathogens the initial event in urinary tract infection

  • Urinary tract infection more common in women than men due to short female urethra with distention and turbulent flow that washes urethral organisms into the bladder during micturition and in close proximity to perianal areas

  • Hospital infection associated with lower urinary tract instrumentation (catheterization, cystoscopy)

  • Once in the bladder uropathogens multiply, then pass up the ureters (especially if vesicoureteral reflux present) to the renal pelvis and parenchyma

  • Source of uropathogens: enteric bacteria

Dr.T.V.Rao MD


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Collecting the urine

  • There are several different methods for collection of a urine sample. The most common is the midstream clean-catch technique. Hands should be washed before beginning. For females, the external genitalia (sex organs) are washed two or three times with a cleansing agent and rinsed with water. In males, the external head of the penis is similarly cleansed and rinsed. The patient is then instructed to begin to urinate, and the urine is collected midstream into a sterile container.

Dr.T.V.Rao MD


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Specimen Collection

  • The urine collected in a wide mouthed container from patients

  • A mid stream specimen is the most ideal for processing

  • Female patients passes urine with a labia separated and mid stream sample is collected

Dr.T.V.Rao MD


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Urine collected with Instructions

Female patients

Male patients

Begin passing urine

Stop flow in midstream

Pass several ml into pen container without touching rim

Stop flow before it ends

Recap container

Pass remaining urine into lavatory

Send specimen to laboratory immediately

(refrigerate if prolonged transport time)

Retract prepuce, using plain soap or antiseptic clean glans. Dry with tissues.

Spread labia,

using plain soap or antiseptic wipe front to back,

dry with tissues

Dr.T.V.Rao MD


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Collection and preservation of Urine Specimens

  • Urine collected in sterile specimen container must be processed within 2 hours, or refrigerated and processed within 24 hours

  • Urine collected in sterile specimen container with borate preservative should be processed within 24 hours (no refrigeration required)

Dr.T.V.Rao MD


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Collecting urine in infants and young children differs from adults

  • In infants, a urinary collection bag (plastic bag with an adhesive seal on one end) is attached over the labia in girls or a boy's penis to collect the specimen.

Dr.T.V.Rao MD


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Specimen collection in infants and young Children adults

  • Non invasive methods are safe and ideal

  • Follow the Broomhall et al method

    By tapping just above the pubis with two fingers place on suprapubic region after 1 hour of feed, tapping on at the rate of 1 tap/second for a period of 1 minute, if not successful tapping is repeated once aging.

    The child spontaneously pass the Urine and to be collected in a sterile container

Dr.T.V.Rao MD


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catheterized urine adults

  • Another method is the catheterized urine specimen in which a lubricated catheter (thin rubber tube) is inserted through the urethra (tube-like structure in which urine is expelled from the bladder) into the bladder. This avoids contamination from the urethra or external genitalia.

Dr.T.V.Rao MD


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Specimen can be collected after clamping the catheter adults

  • If the patient already has a urinary catheter in place, a urine specimen may be collected by clamping the tubing below the collection port and using a sterile needle and syringe to obtain the urine sample; urine cannot be taken from the drainage bag, as it is not fresh and has had an opportunity to grow bacteria at room temperatur

Dr.T.V.Rao MD


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Suprapubic puncture a technically demanding method adults

  • On rare occasions, the health care provider may collect a urine sample by inserting a needle directly into the bladder (suprapubic tap) and draining the urine; this method is used only when a sample is needed quickly and technically competent staff are available

Dr.T.V.Rao MD


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Sending the specimen to Laboratory adults

  • If delivery of the urine specimen to the laboratory within one hour of collection is not possible, it should be refrigerated. The health care provider should be informed of any antibiotics currently or recently taken.

Dr.T.V.Rao MD


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Diagnosis of Urinary Tract Infection adults

  • Step 1

    Microscopy of Urine for detection of Pyuria, Leucocytes should be found in numbers of at least as great as 104 / ml before the pyuria is established

Dr.T.V.Rao MD


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Wet Film examination of Urine adults

  • All wet films to be examined with high power ( x 40 ) objective.

  • Prepare the drop of urine after mixing the urine without centrifugation

  • Transfer 0.05 ml on the middle of the microscope slide and cover slip is applied.

  • The prepared specimen show a small excess of fluid along the edges of the cover slip.

  • A approximate finding of 1 leukocyte / 7 high power fields corresponds to presence of pyuria.

Dr.T.V.Rao MD


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Inoculating the Culture plate with urine adults

  • Plate: provide large surface for isolation and observation of colonies

  • Using a sterile loop or a sterile swab streak your sample on the petri plate

  • Important let your sterilized loop cool before you pick up your sample



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Specimen Inoculations adults

  • All cultures processed by Semiquantitative method a loop of standard dimension of approximately known volume is inoculated into selected culture plate

  • In general a loop of SWG – 28 with a diameter of 3.26 mm internal diameter which can hold a drop of water or urine 0.004 ml.

  • After inoculation the culture plates are incubated at 370c extending to > 18 hours are read

  • The colony counts are made, as each colony corropsdes to number of viable bacteria per ml of urine

Dr.T.V.Rao MD


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Culturing of urine for Isolation of Bacterial pathogens adults

  • Semiquantitative culture

    Select the Media

    For common isolates

    Mac Conkey’s agar

    helps in differentiation of Lactose fermenting organisms from non lactose fermenting pathogens

Dr.T.V.Rao MD


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Choosing media to suit Microorganisms is important adults

  • MacConkey agar utilized as selective differential agar for gram-negative bacteria,

  • Colistin nalidixic acid agar as selective agar for gram-positive bacteria, and chocolate agar for fastidious gram-negative bacteria (Haemophilus)

Dr.T.V.Rao MD


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Inoculation of Urine adults

  • Inoculation of urine for quantitative culture (colony forming units→cfu’s) performed with a calibrated 0.001 mL and 0.01 mL plastic or wire loop

  • Sheep blood agar (SBA) utilized for quantitative urine culture

Dr.T.V.Rao MD


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Culture Media for isolations adultsCLED Medium

It is also an excellent universal culture medium owing to its wide spectrum of nutrients, lack of inhibitors and the fact that it allows a certain degree or differentiation between the colonies. It contains lactose as a reactive compound which, when degraded to acid, causes bromothymol blue to change its colour to yellow. Alkalinization produces a deep blue colouration. The lack of electrolytes suppresses the swarming of Proteus Laboratories which have difficulty in availability of blood agar, CLED medium is opted

Dr.T.V.Rao MD


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Enriched Culture Media for Isolation adults

  • Blood agar

    helps in isolation of fastidious, extracting strains May extended incubation for isolation of pathogens for more than 48 hours with added atmosphere of 5 – 10 % co2

Dr.T.V.Rao MD


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Reading the Culture Plates adults

A true infection in the absence of prior antibiotic therapy the number of bacteria is likely to be at least 105or more.

Contaminated specimens present with colony counts <104, however even less than 103

On several occasions the colonies are diverse species

Several studies prove counts >104 to be considered as presence of Urinary tract infection with the supporting clinical history

On some occasions more than one pathogen is isolated but should be processed for all practical purposes

eg E.coli along with Streptococcus fecalis

On few occasions even counts 103 are proved significant

Dr.T.V.Rao MD


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Identification of Gram + organisms adults

  • All colonies identified morphologically as Staphylococcus to be characterized as

    Staphylococcus aureus

    Staphylococcus saprophyticus

    Staphylococcus epidermidis

    Enterococci - fecal group of organisms

Dr.T.V.Rao MD


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Counting the colonies adults

  • With 0.001 ml loop, 1 colony on SBA equivalent to 1,000 cfu’s per mL of urine

  • With 0.01 ml loop, 1 colony on SBA equivalent to 100 cfu’s per mL of urine

Dr.T.V.Rao MD


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Interpretation of Enterobacteriaceae adults

  • A single species of Enterobacteriaceaerecovered at >105 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 95% probability of true bacteriuria

  • A single species of Enterobacteriaceae recovered at 104-105 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 33% probability of true bacteriuria

Dr.T.V.Rao MD


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Interpretation of Enterobacteriaceae adults

  • A single species of Enterobacteriaceaerecovered at >105 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 95% probability of true bacteriuria

  • A single species of Enterobacteriaceae recovered at 104-105 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 33% probability of true bacteriuria

Dr.T.V.Rao MD


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What is adultsSignificant bacteriuria

  • Significant bacteriuria in an asymptomatic patient is 100,000 or more colonies per milliliter of urine from a midstream, clean-catch specimen; yet, a colony count of 200 Escherichia coli per ml may be significant in a midstream male void or catheterized female. About 95% of all positive UTI cultures will produce essentially pure cultures if urine is collected carefully and the media inoculated promptly.

Dr.T.V.Rao MD


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Up to 10 adults4/ml considered normal i.e. Insignificant

105/ml and above considered to be Significant

Concept valid only for voided specimen of urine

Exceptions - slow growing organisms, patient on antibiotic therapy, diuretic therapy

Concept of Significant Bacteriuria

Dr.T.V.Rao MD


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What can be a significant count adults

  • A single species of Enterobacteriaceae recovered at 104-105 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 33% probability of true bacteriuria

Dr.T.V.Rao MD


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Gram positives and fungi the counts may be <10 adults5

  • Gram-positive, fungal, and fastidious uropathogens often present in lower numbers (104-105 cfu’s/mL urine)

  • Urethral commensals recovered at <104 cfu’s/mL urine

Dr.T.V.Rao MD


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Identification of Isolates adultsGram + isolates

  • The minimal tests to differentiate Gram + cocci include

    1 Catalase

    2 Coagulase test

    3 Bile esculin testing

    4 Bacitracin in Streptococcus isolates

Dr.T.V.Rao MD


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Biochemical tests in Gram - ve bacilli adults

  • Catalase test

  • Oxidase test

  • Nitrite reduction test

  • Indole test

  • Methyl red test

  • V P test

  • Citrate test

  • Decarboxylation tests

    Lysine, ornithine, Arginine tests

Dr.T.V.Rao MD



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Cumitech Guidelines adults for Inoculation of Urine Cultures1

  • Routine: uncomplicated urinary tract infection in ambulatory outpatients (0.001 mL loop, SBA, MAC; 24 hr. incubation)

  • Surveillance: neurogenic bladder, indwelling catheter, geriatric patents (0.001 mL loop, SBA, MAC, CNA; 24 hr. incubation)

  • Special: suprapubic aspirates or straight catheter specimens where previous cultures negative, unresponsive to therapy, or possibility of unusual urinary tract pathogen (0.001 and 0.01 mL loop, BA, MAC, CHOC; minimum 48 hr. incubation

  • 1Clarridge, Johnson, Pezzlo, and Weissfeld, ASM Cumitech 2B, November 1998.

Dr.T.V.Rao MD


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Cumitech Guidelines adults for Interpretation of Routine Urine Cultures1

  • One isolate at >104: Full ID and Susceptibility

  • One or two gram-negative isolates at >105 and other isolates at least 10X less: Full ID and Susceptibility of gram-negative isolates

Dr.T.V.Rao MD


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Cumitech Guidelines adultsfor Interpretation of Routine Urine Cultures1

  • Other patterns of isolates at >104: Presumptive ID only

  • Ignore mixed urethral flora at <104

  • 1Clarridge, Johnson, Pezzlo, and Weissfeld, ASM Cumitech 2B, November 1998.

Dr.T.V.Rao MD


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Cumitech Guidelines adultsfor Interpretation of Special or uncommon Urine Cultures1

  • One or two isolates at >102 to 105: Full ID and Susceptibility is essential before confirmed as uncommon isolates

Dr.T.V.Rao MD


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ASM Manual Guidelines for Urine Culture Results Likely to Be Significant1

  • Midstream, female with cystitis, >102 with positive urine leukocyte esterase

  • Midstream, female with pyelonephritis, >105 with positive urine leukocyte esterase

  • Midstream, asymptomatic, >105 with negative urine leukocyte esterase (usually)

  • Midstream, male with UTI: >103 with leukocyte with urine leukocyte esterase positive

  • Straight catheter: >102 with urine leukocyte esterase positive

  • Indwelling catheter: >103 with urine leukocyte esterase positive or negative

    1Manual of Clinical Microbiology, 8th Edition, ASM, 2003

Dr.T.V.Rao MD


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NMH Guidelines for Interpretation of Urine Cultures Significant1

  • Urine leukocyte esterase positive

    One or two organisms at >103: Full ID and Susceptibility

    One organism at >104 with others (2 or more) at least 10X less: Full ID and susceptibility of predominant organism

    Report all group B β-hemolytic streptococci for women < 50 years

    1Modified from ASM Cumitech, ASM Manual, and CDC MMWR 2002;51 (RR-11):1-22

Dr.T.V.Rao MD


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NMH Guidelines Significantfor Interpretation of Urine Cultures1

  • Urine leukocyte esterase negative

    One or two organisms at >105: Full and Susceptibility

    One gram-negative organism (pure culture) at >104: Full ID and Susceptibility

    Yeast in pure culture: ID as Candida albicans or not C. albicans

    Report all group B β-hemolytic streptococci for women <50 years

    1Modified from ASM Cumitech, ASM Manual, and CDC MMWR 2002;51 (RR-11):1-22

Dr.T.V.Rao MD


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Antibiotic Sensitivity Testing Significant

  • All the isolated bacteria identified as pathogenic to be tested for Antibiotic Sensitivity/Resistancepattern by disk diffusion methods

Dr.T.V.Rao MD


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Antibiotic Sensitivity Significant

  • Always use a pure growth of the isolate.

  • Perform testing for antibiotic sensitivity with inoculum which is comparable to defined McFarland standards

Dr.T.V.Rao MD


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Antibiotic Sensitivity Testing Significant

  • High potency disks used

  • Antibiotics normally active in urine selected

  • Antibiotics found in higher concentration in urine preferred

  • Primary sensitivity test frequently used

Dr.T.V.Rao MD


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Reporting of Contaminated specimens Significant

  • Microscopy helps to detect pus cells and epithelial cells, an insignificant growth with few pus cells can ignored as contaminants

  • In females even the presence of leucocytes with Squamous epithelial cells without a defined significant growth should be ignored.

Dr.T.V.Rao MD


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Reporting of negative urine cultures Significant

  • Negative culture results showing no bacterial growth are available after 24 hours. Positive results require 24-72 hours to complete identification of the number and type of bacteria found

Dr.T.V.Rao MD


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Clinical Problems manifesting as Urinary tract Infections Significant

  • An infection of the Genitourinary tract where the Microbes Donot grow on selected media as in Mycobacterium tuberculosis

    Gonococcus

    Nutritionally exacting or anaerobic bacteria

    should be considered.

Dr.T.V.Rao MD


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Urinary Significanttract infections are over diagnosed

  • Most over diagnosed condition, both by the clinicians and microbiologists.

  • Makes the physicians to miss the existing condition.

  • Unnecessary antibiotic prescription lead to drug resistance, loss of resources

Dr.T.V.Rao MD


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  • Created by Dr.T.V.Rao MD for ‘eLearning” resources for Microbiologists in the Developing World

  • Email

  • [email protected]

Dr.T.V.Rao MD


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