Microbiology Laboratory



Microbiology Laboratory



Objectives


At the end of this chapter, the reader should be able to do the following:


1. Calculate the colony forming units or concentration of bacteria per milliliter of sample in a urine specimen when using a calibrated loop.


2. Correlate the quantity of colony forming units from a urine culture with probability of urinary tract infection.



COLONY COUNTS


A colony count determines the amount of bacteria present in a sample. The colony count is often used in the microbiology laboratory to determine the amount of bacteria present in a urine sample. Urine is a sterile fluid formed by the kidney and stored in the bladder until voided. As urine passes from the bladder into the urethra, it may become contaminated with the normal flora found in the urethra. Bacteria that is on the skin in the genital area may also contaminate the urine specimen.


The total amount of bacteria present as a result of contamination is less than 10,000 organisms per milliliter of urine or less than 10,000 or 103 colony forming units (CFUs). Urinary tract infections are primarily caused by single strains of bacteria. Urinary tract infections that are caused by more than one strain of organism are not very common. If three strains of organisms are detected, then contamination of the specimen has most likely occurred. The most common organism responsible for urinary tract infections is Escherichia coli, a gram-negative rod.


When a physician suspects that a patient has a urinary tract infection, a clean-catch urine sample is collected by the patient. When the concentration of organisms is greater than 100,000 organisms per milliliter (105 CFU per mL), a urinary tract infection is indicated. If a urine sample is to be used for both microbiologic studies and urinalysis, the microbiologic studies are performed first to avoid contamination of the sample with reagent strips, pipettes, and so forth.


Although there are other methods to perform a colony count, the streak method will be discussed in this chapter. In the streak method, a calibrated loop is used to inoculate a culture plate. Two different calibrated loops can be used. One calibrated loop will hold 0.01 mL of sample. The other loop will hold 0.001 mL of sample. The urine is streaked first with a primary streak down the middle of the plate. The plate is then turned 90° and streaked across the primary streak the length of the plate. In some labs, the plate is again turned 90° (or 180° from the original orientation) and streaked across the secondary streaks. In this manner, the entire plate is streaked with urine, and isolation of any organisms can be achieved.


The plate is incubated aerobically at 37°C for 24 hours, and the number of colonies or CFUs per mL are counted. Each CFU per mL represents a single organism from the original sample. The total number of CFUs per mL are calculated by multiplying the amount of colonies by the dilution factor of the calibrated loop. If a 0.01-mL calibrated loop is used, the number of colonies is multiplied by 100. If a 0.001-mL calibrated loop is used, the number of colonies counted is multiplied by 1000. In this manner, the total number of CFUs per milliliter of urine can be obtained. Clean-catch urine specimens that contain less than 103 CFUs of bacteria are not indicative of a urinary tract infection. Clean-catch urine specimens that contain between 103 and 105 CFU may indicate possible infection.


When a possible infection is indicated, the organism is identified, and antibiotic susceptibility testing is performed. Clean-catch urine specimens that contain more than 105 CFUs per mL indicate probable infection. As with a possible infection, the organism is identified and antibiotic susceptibility testing is performed.


Urine may also be collected from an indwelling catheter. Patients with indwelling catheters are at high risk for acquiring urinary tract infections; therefore, sterile technique should be carefully followed. Urine is never collected for culture from the collection bag. Significant bacteriuria is indicated if greater than 105 CFUs per mL are counted.


Urine may also be collected by suprapubic aspiration. In this surgical procedure, urine is taken directly from the bladder and cultured. Any amount of CFUs per mL should be reported, the organism identified, and antibiotic susceptibility testing performed.






Example 11–1

A clean-catch urine specimen is obtained from a 17-year-old girl with a possible urinary tract infection. The urine is cultured using a 0.01-mL calibrated loop. After appropriate incubation, 350 colonies are counted. What is the patient’s CFU/mL, and is it indicative of a urinary tract infection?


A 0.01-mL calibrated loop was used to inoculate this urine. Therefore the quantity of colonies, 350, is multiplied by the dilution factor of 100, yielding 35,000 CFU/mL.


A CFU count of at least 105 colonies is necessary to indicate probable infection. The patient’s CFU/mL count was 3.5 × 104, indicating possible, but not probable, infection.



Additional Examples




image A clean-catch urine specimen was obtained from a 42-year-old woman with a possible urinary tract infection. The urine was cultured using a 0.01-mL calibrated loop. After appropriate incubation, 8 colonies are counted. What is the patient’s CFU/mL, and is it indicative of a urinary tract infection?


Since the 0.01-mL loop was used, the number of colonies, 8, is multiplied by 100, yielding 800 CFU/mL, or 8.0 × 102 CFU/mL. Since this is less than 103 CFUs of bacteria, the result is not indicative of a urinary tract infection.


image A clean-catch urine specimen was obtained from a 12-month-old boy with a possible urinary tract infection. The urine was cultured using a 0.01-mL calibrated loop. After appropriate incubation, 1245 colonies are counted. What is the patient’s CFU/mL, and is it indicative of a urinary tract infection?


Since the 0.01-mL loop was used, the number of colonies, 1245, is multiplied by 100, yielding 124,500 CFU/mL, or 1.245 × 105 CFU/mL. Since this is more than 105 CFUs of bacteria, the result is indicative of a probable urinary tract infection.

Related posts:

  1. Quality Assurance and Quality Control in the Clinical Laboratory
  2. Urinalysis Laboratory
  3. Dilutions and Titers
  4. Calculations Associated with Solutions

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Nov 18, 2017 | Posted by in PHARMACY | Comments Off on Microbiology Laboratory

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