Microbial monitoring with serial dilution culture media

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 serial dilution bottle tests highly inaccurate. However, in the early days of microbiologically influenced corrosion (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. 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 color, 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 recipe of the 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.

Molecular methods such as the ATP test bypass the need for culturing and the complications that arise from that, making them far more accurate. The ATP results are also available within minutes, compared to weeks for the culture tests.

With a serial dilution, you take a sample and create a dilution series and then allow the bottles to incubate for up to 28 days (for SRB). The number of bottles that change color represents the log number of microbes (ie if you prepare 6 dilution bottles and the first 3 change color, then you have 103 microbes/mL in your sample).

A BART test (biological activity reaction test) has desiccated media at the bottom of a tube with a ball on top. You add your liquid sample and let the tube stand still for about 10 days. The ball on top allows some oxygen in, and you end up with an oxygen gradient that allows aerobes to grow at the top and anaerobes to grow at the bottom. Microbial growth produces color changes at different levels along the gradient, which can be roughly quantitated in a similar fashion as the dilution tests. Both tests require a liquid sample, however solids can be done if they are first re-suspended in a liquid buffer. BART tests can detect more microbial groups, such as iron related bacteria, however in the end both dilutions and BART tests are growth based methods, which has its drawbacks.

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 native 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.

As explained above, culturing microbes is very difficult and will give an underestimation of what is in a sample. So, when you compare a growth test to a molecular test that does not require culturing, the molecular test will always have a higher result.