LifeCheck Microbial Service Resources
Glossary of LifeCheck Microbial Service Terms
|16S||16S is an rRNA gene found universally in all microbes (it encodes for highly important protein building machinery) that is analogous to a fingerprint – it can be used to identify the microbe(s) down to the species level. The 16S gene can be used in several molecular MIC tests.|
|Archaea||A domain of microorganisms (microbes) very similar, yet distinct from bacteria.|
|ATP||Adenosine Triphosphate or ATP is the energy currency of cells. All cells (microbe or even human) use energy in the form of ATP to do work.|
|Bacteria||Single cell, microscopic organisms. Bacteria are found everywhere (capable of living under a very wide range of habitats including extreme environments).|
|Gene||A functional region/unit of DNA within an organism (microbe). Genes have codes to make proteins that provide a specific function to the microbe. Example, the dsrAB gene(s) codes for proteins that are used in sulfate reduction by SRB.|
|Metagenomics||Sequencing all of the 16S genes (reading all of the fingerprints) in a sample gives a list of all the microbes present, along with relative abundance percentages (semi-quantitative assay).|
|MIC||Microbiologically Influenced Corrosion (MIC) describes the corrosive damage to surfaces caused by microbes, including bacteria and archaea.|
|Microbe||A unifying term used to describe bacteria and archaea (microscopic, unicellular organisms).|
|MMM||Molecular Microbiological Methods (MMM) are culture-independent, genetic-based assays for MIC diagnostics.|
|qPCR||Quantitative Polymerase Chain Reaction (qPCR) is a molecular microbiological method (MMM) that functions by counting/enumerating instances of a gene of interest in a given sample. For example, by counting the number of 16S genes in a sample, one can quantify the total number of microbes.|
LifeCheck Frequently Asked Questions
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.
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 colour represents the log number of microbes (ie if you prepare 6 dilution bottles and the first 3 change colour, 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 colour changes at different levels along the gradient, which can be roughly quantitated based on rate of change. Both tests require a liquid sample, however solids can be done if they are first resuspended in a liquid buffer. BART tests can detect microbial groups such as iron related bacteria, however in the end both dilutions and BART tests are growth based methods, which as we have discussed 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 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.
Emulsions or crosslinked frac fluids are very viscous and difficult to pass through the filter in the ATP procedure. When getting a few mL’s through is hard to do, we have a modified procedure you can use. See the reference section for PART A4: SAMPLE PREP – Modified Soak Procedure for Viscous Fluids. In this procedure the viscous fluid is placed in a soak reagent and agitated in order to access the microbes in the sample. There is a separate calculation in order to accommodate this modified procedure.
Microbes can only survive in water, so when you are testing an emulsion, be aware that the microbial count is based on the water cut in that sample, and you may want to calculate based on water content rather than total fluid volume.
The calculation used to convert pg of ATP/mL into ME/mL is based on the amount of ATP present in an E.coli microbe grown in the lab, which is 0.001pg. This is a reasonable value to use based on extensive research on different microbe species. It is noted in many literature sources that the average ATP in a human is 250g, but we know this will vary person to person. Microbes, like humans, come in all shapes and sizes and levels of activity, meaning ATP will vary in the same way. However, we can choose a reasonable value to convert pg to a more easily understood unit of measure for industrial applications.
Always remember that although you may have comparable units, what you are measuring changes with each test method, and often direct comparisons are not appropriate.