Module 9: Urine Culture and Sensitivity

Module 9.4: Enumeration of Bacteria

As we learned earlier, the bladder is not a sterile place, but it generally contains such low biomass of bacteria it is generally unculturable.

How can we determine the number of organisms cultured in the urine?

For urine, we inoculate a measured amount of urine onto a non-selective (differential) plate such as the BAP, to determine the colony-forming units (CFU) present in the sample.

How do we calculate the CFU’s in a sample?

A is defined as a bacterial cell or cluster of cells that give rise to a colony on a plate.

BAP with many CFU's
BAP with many CFU’s

On the entire BAP, each individual colony or cluster is recorded and then entered into the following equation. You will be practicing this in the lab.

 

CFU (per mL) Equation

CFU (per\:mL)=\frac{(\#\:colonies\:present\:on\:the\:plate)(dilution\:factor)}{volume\:plated\:(mL)}

 

By counting the colonies, you can determine the concentration of bacteria in the original urine sample.

 

Case Example Calculation

Things we know: 

  • The sample was collected via cystocentesis
  • The loops that you will use in the lab are 1μl (volume plated)
  • The number of colonies you counted on the plate was 50

Let’s get started:

The first step is to convert μl to ml 1μl → is equal to 0.001 ml

Your urine was not diluted and applied directed from the sample collected from the animal, the dilution factor is 1.

So if we plug those values into the equation….

50\times10^{3}\:CFU\:per\:mL=\frac{(50\:colonies)(1)}{0.001\:mL}

 

Your patient has 50,000 CFU/ mL of bacteria.

Clinical question: Does this patient have a UTI from the CFU/mL calculated?

How can we determine if the number of organisms cultured is abnormal?

Based on the work of Edward Kass in the 1950’s evaluating UTIs in women, we use the same cutoffs to define “too much bacteria” in the urine as the human medical profession.

The dividing line between disease and no disease is the following:

Cystocentesis collection: ≥ 103 CFU/ml

Catheterized specimens, counts ≥ 104 in males and ≥ 105 CFU/mL in females is significant.

*Per ISCAID, voided samples should never be used for culture. However if a voided sample is the only option ISAID states, “Culture of voided samples should only be performed when cystocentesis is contraindicated because of the potential for both false positive and false negative cultures. Voided samples should only be cultured if they are refrigerated and processed by the diagnostic laboratory within a few hours or cultured in-house (Sørensen et al., 2016). The level of growth (≥100,000 colony forming units (CFU)/mL), bacterial species (i.e. isolation of common uropathogens such as Enterobacteriaceae or coagulase-positive staphylococci) and whether pure growth is present are important factors to assess when evaluating culture results from voided samples, along with urine cytology and clinical signs. Laboratories should be informed whether urine samples are cystocentesis or voided samples, to ensure that quantitative culture is performed on voided samples.”

So for the patient above, culturing 50,000 CFU/ ml is too many. If the animal has clinical signs of UTI, then these results support the clinical diagnosis of UTI. The diagnosis of any disease using culture is should not be extrapolated from culture data alone. The bacterial culture data is used to support your diagnosis.


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