Frequently Asked Questions

How do LifeCheck ATP Test Kits work with produced waters?

The steps of the ATP test are quite simple. First, a sample is taken in a syringe and passed through a filter to remove any debris and trap the cells on the filter. This is followed by a washing step to remove any contaminants and another wash step to lyse the cells. The solution collected after the final wash (lysing) contains the ATP, and an enzyme is added which causes the ATP to emit light which is then measured using the LifeCheck PhotonMaster. The entire test takes about 10 minutes.

Can LifeCheck ATP Test Kits work with solid samples?

Yes, for solids samples we have the sessile kit which has an extra step of mixing the solid with a buffer solution to create a liquid suspension.

What does the result from the LifeCheck ATP test tell us?

The result gives us the amount of total living cells, or how much ‘life’ is in your sample. It is essentially the result of combining all other traditional growth tests into one, with the added benefit of being far more accurate as no culturing step is required. It is an easy and accurate method to assess biocide treatments and efficiency, and for routine microbial monitoring.

How does the test results from LifeCheck ATP compare to traditional culture media like APB/SRB?

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.

What is the difference between serial dilution tests and BART test?

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 resuspended 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.

Why is it difficult to grow bacteria?

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.

Why can there be such a difference between logs of ATP and MPN log of bacteria?

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.

Are there other methods to differentiate the types of bacteria?

Culturing provides a general distinction between microbial metabolisms, but does not provide meaningful information. This is because most microbes can carry out multiple functions depending on the environment, eg. SRB can produce acetate, which would also make them an APB. Molecular methods (refer to nace tm0212) are far superior at determining which groups are present and their abundance, and they also bypass the troubles related to culturing.

Alternative tests include ATP, FISH (fluorescent in-situ hybridization), DAPI (4′,6-diamidino-2-phenylindole), qPCR (quantitative polymerase chain reaction) and metagenomics/16s sequencing. With ATP, you can get a snapshot of the total microbes present in your sample, regardless of the species or type. FISH, DAPI and qPCR are useful for accurately measuring abundance, however you need to first know what you are looking for, as specific probes are required for each microbial group or species you wish to detect. 16S sequencing will tell you all species which are present in a sample, however to quantify, one of the other molecular methods is required in conjunction with the sequencing.

These additional molecular methods do require a lab and a skilled technician to carry them out, which is a drawback. For this reason, OSP launched LifeCheck Resources.

What are the ATP Test Kit storage requirements and conditions?

When you receive your test kit, utilize the following guidelines for material storage. Avoid usage of expired test kit components.

  1. Reagent Z and Buffer
    • Recommended Storage: 4 to 25°C / 39 to 77°F
    • Shelf Life: 2 years
  2. Reagent Z Liquid
    • Recommended Storage: 4 to 25°C / 39 to 77°F
    • Shelf Life: Note Below
  3. All Other Reagents (X,D, Standard, Rinse and Soak)
    • Recommended Storage: 4 to 25°C / 39 to 77°F
    • Shelf Life: 2 years
What are the long-term production benefits of treating with 2K7 biocide?

The usage of 2K7 biocide prevents bacterial contamination and growth, controlling issues that arise from their presence, including microbiologically influenced corrosion (MIC). 2K7 reduces the risk of failures, improves run time on pipe, prevents contamination of downhole equipment and controls formation damage.

What is ME/ml?

It is a conversion from pg/ml to microbial equivalents so you can compare the results to other technology readings.

General Contamination Guidelines for Oil and Gas Applications (Planktonic ATP Levels)
LowModerateHigh
< 100 pg/mL100 – 999 pg/mL≥ 1,000 pg/mL
< 105 ME / ml105 ME / ml≥ 106 ME / ml

Note the results must be taken into consideration with what you see and what the current problem is. The above is a guideline that needs to be adapted to your system. For example, a moderate level of microbes may cause souring in one system, but no issues at all in another. Microbes don’t have a linear relationship to microbial issues, therefore there’s no exact answer for contamination levels. This is simply a baseline to work from.

How do ATP readings compare to bug bottle readings?
THREATME/mlBottle TurnsATP Reading
OXIDIZER LOW0 - 9.99 x 103 ME/ml0 - 31 - 10 pg/ml of ATP
LOW1 x 104 ME/ml - 5 x 104 ME/ml410 - 50 pg/ml of ATP
MODERATE5 x 104 ME/ml - 1 x 105 ME/ml550 - 100 pg/ml of ATP
HIGH1 x 105 ME/ml - 1 x 106 ME/ml6100 - 1,000 pg/ml of ATP
VERY HIGH> 1 x 106 ME/ml6+> 1,000 pg/ml of ATP

Note the results must be taken into consideration with what you see and what the current problem is. The above is a guideline that needs to be adapted to your system. For example, a moderate level of microbes may cause souring in one system, but no issues at all in another. Microbes don’t have a linear relationship to microbial issues, therefore there’s no exact answer for contamination levels. This is simply a baseline to work from.

How do microbes contribute to corrosion?

The reason MIC is defined as “influenced” is because the bacteria don’t directly corrode the metal, but they accelerate the process by affecting the chemical reactions that cause corrosion. Biofilms – layers of bacteria in a matrix of slime – grow on metal surfaces changing the physical and chemical microenvironment. For example, anaerobic sulfate reducing bacteria (SRB) convert sulfate into hydrogen sulfide – a strong acid. This effectively creates a mini battery, with the metal pipe providing the negative and positive poles (anode and cathode). The resulting
localized chemical reactions dissolve the metal within a small area – increasing the chances of metal failure.

Other types of bacteria have been implicated in MIC, such as acid producing bacteria (APB) and metal reducing or oxidizing bacteria (MRB and MOB); however, more research is needed.

Why is ATP monitoring effective in establishing a treatment baseline?

ATP monitoring offers a powerful combination of speed, versatility, portability, and accuracy for microbial testing. All living cells contain ATP regardless of whether they are bacteria, fungi, or any other type of microbe. As such, its measurement is a direct indication of the microbial content in your sample. Our LifeCheck ATP test kit is an easy, fast and accurate microbial enumeration method, valuable in routine monitoring and supporting microbial management programs.

What is archaea and can it contribute to MIC?

Archaea are a domain of microbes/microorganisms, very similar, yet distinct from bacteria. A number of species have been identified that contribute to MIC within this domain. That being said, the behavior is similar and our control methods are employed in the same way.

Within an ATP Test, is the accuracy affected based on the volume pushed?

Increased volume or sample size provides a more accurate average of microbial load per ml. There is increased sensitivity when you have a low sample volume. The rule of thumb though is to be consistent within comparable samples.

Do you freeze or refrigerate enzymes?

When you receive the Reagent Z and Buffer, keep frozen until mixing for use. Once you mix and the enzyme is in liquid form, refrigerate.

Is 2K7 compatible with most chemistries?

Yes. 2K7 is non-ionic and therefore very compatible with most system components within the oil and gas industry.

Can microbes develop a resistance to 2K7 over time?

Very unlikely. While it has been proposed that microbes can develop resistances to organic biocides, this has not been seen with 2K7 applications nor is it likely based on application methods used within our industry.