What Do Microbiology Test Results Really Mean?

Microbial stress and survival are other factors that can affect test results. In dry, nutrient-poor environments microbial viability declines rapidly in a matter of hours such that there is a large variation in observed contamination levels. Literature shows examples of total counts <2 to 5.0 x 105 CFU/centimeter (cm)2 with E. coli detected on 15.8 percent of the samples with a range of 0.2 to 12 CFU/cm2.

Inoculating surfaces is known to result in large losses of viability with hugely variable residual contamination levels. Inoculating surfaces with a suspension containing 1 million bacteria can give a final residual contamination of 10 to 100 bacteria with 100 to 500 fold variation between five replicates from the same inoculum. Re-suspending and recovering contaminants from the surface swab into a diluent prior to testing also introduces another source of variation. Therefore, great care needs to be exercised when assessing the results of environmental tests and also when comparing methods for the assessment of environmental contamination. Accordingly, the enumeration of microbes in environmental samples yields little meaningful information. A qualitative (presence/absence) approach is more appropriate. General guidelines have been suggested by some authors and auditors, e.g. acceptable ≤80 CFU/cm2 and unacceptable ≥1,000 CFU/cm2. Trend analysis is more suitable and gives better management information about risks and emerging problems. The benefits of regular testing, preferably with a simple method giving rapid results for prompt corrective actions, are well established.

Alternative Rapid Method

An example of a new test method that uses traditional microbiology with an end detection system is Hygiena’s MicroSnap. This two-step test procedure has a total time to result of 7 to 8 hours. The sample can be a surface swab, a 1 milliliter (ml) liquid sample, or a food suspension. Samples are mixed with a proprietary enrichment broth in an all-in-one device, and then incubated for 7 to 8 hours. After incubation, a 0.1 ml aliquot is transferred (using the device itself) to a specific end-detection device and measured with the EnSURE luminometer, which also measures adenosine triphosphate sanitation monitoring tests, among other food quality indicators. MicroSnap is formulated for a variety of bacteria and is currently available for Total Counts, Enterobacteriaceae, coliform, and E. coli.

The output of MicroSnap is directly related to inoculum size, i.e. the greater the number of bacteria the shorter the time to detection. Typical results for Total Counts and Enterobacteriaceae have been known to show excellent agreement and a high coefficient of correlation (>0.90) when compared with traditional plate counts. The dynamic range of the single test device is 10 to 10,000 bacteria per ml (or swab), thus negating the need for serial dilutions. A shorter detection time can be set according to the desired specification. For example, 100 Enterobacteriaceae can be detected in 5 hours.

All viable bacteria collected on the swab are cultured and detected within the system. This permits maximum recovery and minimal losses. A study of 300 surface samples showed an 89 percent agreement with the traditional plate count methods for both Total Counts and Enterobacteriaceae and the limit of detection was calculated as 50 to 100 CFU per swab, or ~1 CFU/cm2. In a small proportion of cases (7 percent), samples were positive when tested with MicroSnap and negative when tested with traditional methods.

In summary, the results of microbiological methods are naturally very variable and must be interpreted with care and recognition of limitations. Pragmatism and practical solutions are required to establish “reasonable expectations” for the results from microbiological methods. Results from environmental samples are subject to even greater variation. Therefore, qualitative measurements and trend analysis can provide the most meaningful information.