Microbial Identification and Evaluation

The ATP test is a simple field test that detects a molecule called Adenosine Triphosphate, or ATP. This molecule is related to energy production and is present in all living cells. The ATP test uses a reaction that generates light when ATP is present, and is easily measured with a simple tool in the field. By measuring the ATP content of a sample, you can assess the viable biomass or “total life” within that sample. This test does not distinguish between microbial groups (SRB, APB etc). Results take less than 10 minutes per sample.

These tests aren’t comparable as they work differently and measure different things. Culture tests are trying to grow microbes, and estimate microbial numbers based on microbial growth. We now know that less than 1% of microbes are culturable even under perfect lab conditions, making this type of test highly inaccurate. However, in the early days of MIC, these were the only tests available and were a positive first step. These tests work by adding a volume of liquid sample to a vial of pre-made culture media and performing serial dilutions to estimate the log numbers of cells in the original sample. The vials must incubate for 3-4 weeks before the results can be interpreted, which is simply a visual assessment of how many bottles turned cloudy or changed colour, depending on the test. Several studies have shown from sequencing the DNA of original samples and incubated samples that the samples have completely different microbial communities. In general, the type of media used determines the numbers and types of microbes you will get.

There is also a high potential for false negatives as many microbes simply cannot grow in the culture media, even ones that are of interest. As a result, you end up underestimating your microbe count. For example, sulfide in a system can come from a variety of microbes other than SRB, such as thiosulfate reducers and Archaea. These other bugs will not be able to grow in the SRB media, meaning that you may get a negative result for an SRB bottle test simply because the actual sulfide producers in your system cannot be detected. This can have significant and dangerous results.

The idea of unculturable microbes first appeared around 100 years ago, when it was observed that cell counts under a microscope were much higher than the number of colonies that would grow on a plate. It has been highlighted again with recent molecular technologies, such as 16S sequencing, which showed us that a single sample can have thousands of different microbes in it.

Think of microbes as tiny people, they have specific conditions required for growth. We cannot survive outside of a narrow temperature range, for example. Microbes also have complex nutrient and environmental requirements. Replicating the conditions required for growth is the biggest challenge, as microbes have diverse pH, nutrient, salinity, temperature, oxygen and pressure requirements.

For example, high temperature microbes, known as thermophiles, will die below a certain temperature, and incubating them at room temperature will kill them. Same with microbes from a high salinity environment, if you put them into culture media with less or different salts than their environment, they will die. In addition, most microbes live in complex communities where they live syntrophically (they depend on each other) with other microbes, and cannot grow efficiently on their own. Think of it this way, if you were picked up and dropped into the middle of the ocean, how long would you survive? You suddenly don’t have food you can eat, or water you can drink even though you are surrounded by water and fish.

In addition, microbes grow in the natural environment which has a relatively low nutrient concentration, and consequently they are slow growers which can take months or even years to replicate. These microbes may be well established in your system, however trying to grow them to measurable amounts would take a considerable amount of time. In some cases, putting microbes from a nutritionally low to high environment can shock the cells to death. Same reason why a starving person cannot just sit down and eat a large meal after months of not eating, they will become very sick.