A very common question that gets asked is: “Why does my ATP test tell me that I have substantial microbial contamination when my culture-based tests tell me that I have little to no contamination? Is the ATP test giving me a false positive?” The terminology ‘false positive’ is very dangerous, and it assumes that the test method in question (in this case, the ATP test) is giving erroneous information. In fact, since the ATP test measures only ATP, and ATP can only come from biological sources, it is not possible to have a ‘false positive’ in the ATP test. The reason that this can happen is due to a lack of understanding of how each test method goes about quantifying microorganisms. This is covered in the following slides. 1

+28moregenerations visiblecolony  10 9 cells X X noreproductionnocolony generation time  4h   5d to visiblecolonygeneration time  1h   1.5d to 1fg active cell -1  1,000 cells pg -1  1CFU mL -1 Every active cell measured! Slow growing & injured active cells missed! active cells missed! [ATP] cell -1  constant;  no growth or metabolism needed after sample collected 2

1. Not all microbes like the same food 2. Different types of microbes have different oxygen needs Obligate aerobes (require oxygen) Obligate anaerobes (can’t tolerate oxygen) Facultative anaerobes (grow with or without oxygen) Under any one set of growth conditions  cell per 1,000 will be detected as a colony 3. Microbes with doubling times >4h won’t be seen until after 5 days incubation; most tests stop at 3 days T G = 0.5hT G = 4h Day 1Day 5 Day 5 a Day 1 Day 5 b 3

The streak plate test was intended for use to obtain pure colony (axenic) cultures. A 10 µL loop-full of sample is streaked across a plate. The assumption is that the break-point (see illustration below) at which continuous growth separates into individual colonies is proportional to CFU/mL. This test is therefore semi-quantitative and not ideal for the enumeration of microorganisms. This is especially true in viscous solutions, where microbiological heterogeneity is much higher, leading to a larger variance in results when taking only a 10 µL sample! Overall, this method will underestimate population size. 4

As is demonstrated in the previous slides, the key difference to understand is what, exactly each of these methods quantify: The ATP test quantifies total active microorganisms. The culture test (in any form) quantifies specific viable microorganisms depending on the parameters of the test method. By recognizing the key differences between these methods, one can put them to action in the best ways and generate the best data possible to achieve process improvement! 5

Viable sub-populations of total population Total microbial cell count ≠≠ The best control strategy is to use a combination of tools to characterize the situations from many points of view. ATP is the first line of defense – complete and fast! Water Quality Gross Observation Conventional Counts 2 nd Gen ATP Estimate of activity of total population 6