Quantitative and Qualitative Microbiological Testing: It is quality and quantity that matter
In the world of microbiology, both quality and quantity matter just as much as in other areas of life. However, there is a specific distinction among microbiological methods with some classified as qualitative and others as quantitative. What do these terms mean, and why does this matter?
What is Qualitative Microbiological Testing?
Qualitative methods detect, observe, or describe some quality or characteristic. In microbiology, qualitative methods are used to observe characteristics of isolated microorganisms allowing us to identify them or, more frequently, to detect the presence of organisms of interest or concern. In food or clinical diagnostic microbiology, the most frequently used qualitative tests are those designed to detect particular organism(s), typically pathogen(s), in a given sample. These tests are usually very sensitive and can detect the target organism(s) even at very low population. Qualitative methods have a limit of detection (LOD) nominally equivalent to 1 CFU (Colony Forming Units) per test portion. Test portions can range in size from 25 g to 375 g or even 1500 g.
Cultural methods or rapid screening methods can all be qualitative when used with no path to direct enumeration. To achieve the required sensitivity, all use an amplification step, traditionally enrichment but various concentration procedures may also be used. Following amplification, the method proceeds to detection. For cultural methods, this involves subculturing from the enrichment to plates of selective and differential media which, following incubation for 24-48 h or more, are examined for colonies typical of the target organism. For rapid screening methods a portion of the enrichment is further processed to allow instrumental detection of cellular components such as cell-surface antigens or target sequences of cellular DNA or RNA within a few hours.
The need for the amplification step to raise the concentration of the target organism to a detectable level breaks the direct link to the concentration of the target in the sample. There are ways to estimate the initial concentration from the time needed to reach detectable concentrations and/or the number of amplification cycles to detection using quantitative PCR (the Ct or Cq value). However, these estimates have very wide confidence intervals and are usually not attempted for that reason.
Microorganisms Detected Using Qualitative Testing Methods
Qualitative tests important in food microbiology include analyses for human pathogens such as Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 or other Shiga-toxigenic E. coli (STEC). Some spoilage organisms that can be problematic when initially present at very low concentration, such as Alicyclobacillus spp. in pasteurized juices, may also be detected using qualitative methods.
Qualitative Laboratory Test Results
The results of qualitative tests can be reported in several different ways including “Negative or Positive,” “Detected or Not Detected,” of "Absent or Present" per the tested weight or volume analyzed – for example, Not detected/25 g or Positive/375 g. For rapid screening methods, a detection event may be reported as e.g., Presumptive/375 g with this result resolving to Detected or Not Detected after an additional cultural confirmation.
What is Quantitative Microbiological Testing?
Quantitative methods measure numerical values. In microbiology, this is often the population size (total numbers) of specified microorganisms in each gram or mL of a sample. There are quantitative tests for microbial indicators such as aerobic plate count, Enterobacteriaceae, and coliforms, and others for specific target organisms such as Staphylococcus aureus, or Bacillus cereus. These are reported as number of organisms per unit weight or volume.
Importance of Serial Dilution for a Countable Range
All methods that involve counting colonies on agar plates specify a “countable range” such as 25 to 250 or 30 to 300 representing the number of colonies allowed to be counted on a plate. This range is statistically determined to reduce variability, thereby improving accuracy, and to avoid interference or competitive inhibition between colonies. To achieve these countable ranges, most samples will be diluted in a series of 10-fold steps and a range of dilutions will be plated to increase the chance that at least one dilution will fall in the countable range. It follows that accurate and precise dilutions are critical.
Detection Limits of Quantitative Methods
Quantitative “plate count” methods usually have a limit of detection (LOD) of 10 or even 100 CFU/g and most probable number (MPN) methods typically have LOD of 3 MPN/g. Hence, it is quite possible to detect a pathogen with a qualitative method but be unable to count it. However, if a quantitative method can give a count of an organism of interest, we can be sure that organism is present.
If no growth of the target organism(s) is detected, the result will be reported as “less than” the limit of detection (i.e., <10 CFU/g or < 3 MPN/g). Often people ask “Is <10 CFU/g the same as a negative result?” The short answer is no, they are not the same. Avoid the sensitivity trap!
Reported Values and Quantitative Reporting Terminology
The final count is typically calculated as a colony forming unit per gram, serving, or other unit of measure from the number of colonies grown on the countable dilution multiplied by the dilution factor. For example, if there is one colony on the plate and 1 ml of the 1/10 dilution was plated, the result is 10 CFU/g of sample. If only 100 µl were plated the result would be 100 CFU/ml.
When no bacteria are recovered on the plate, the method specifies that a plate with no colonies at the lowest dilution shall be reported as < LOD for that dilution.
There are times when only colonies from one dilution can be counted, and the number is above or below the countable range. In these cases, the result from the count and dilution is calculated as normal, and then is labeled with “est.” to show that the result is an estimate from an “out of range” count.
Qualitative vs. Quantitative Method Selection
Whether to select a qualitative or a quantitative method depends on the goals of the analysis. If the need is to detect 1 pathogen CFU in a large portion of sample, then a qualitative method sensitive to that pathogen is needed. If the purpose is to discover the concentration of specified microorganisms in the sample, then quantitative methods selective for the organisms of interest are needed.
For further questions on method considerations specific to your process or results: