Frequently Asked Questions

ATP monitoring offers a powerful combination of speed, versatility, portability, and accuracy for microbial testing. This testing measures the total active/living microbial load (count) in the sample. It is an ideal form of testing for creating a baseline, and for building trend data to ensure that an asset is being well maintained. For example, ATP testing is used to evaluate the effectiveness of the kill achieved through microbial control programs. Biocides used in oil and gas are non-specific – they don’t preferentially kill one type of microbe over another. Reading the total microbial load before and after a treatment program mitigates against unexpected outcomes.

ATP takes approximately 5-10 minutes to complete and can be conducted on-the-go (portable) or in a lab. For more information, watch our training videos or see our LifeCheck ATP Handbook (contact us for details).

The entire test takes 5-10 minutes, briefly the procedure involves:

1. Drawing the sample into the supplied syringe and passing it through a filter to remove any debris and trap the cells on the filter
2. A rinsing step to remove contaminants
3. Passing the Reagent X through the filter to break open the cells, releasing the ATP for analysis
4. The solution collected after passing Reagent X through the filter contains the ATP
5. Mixing the resulting liquid with Reagent Z that contains an enzyme that uses this ATP to emit light
6. Measure the amount of light (RLU’s) using the LifeCheck PhotonMaster
7. An easy to use calculator tool then converts the RLU reading to a measure of microbial load (Microbial Equivalents)

For more information, watch our training videos or read our ATP Handbook (contact us for details).

Increased volume or sample size provides a more accurate average of microbial load per unit. Decreasing sample volume may decrease assay sensitivity, especially in low microbe samples. The rule of thumb though is to be consistent within comparable samples. Aim to sample (filter) 10-60 mL.

Yes, for solids samples we have the sessile kit, specifically designed for testing microbial loads on solid and surface (swab) samples. Sessile testing refers to testing for surface-attached microbes, such as those that cause corrosion-related failures of assets.

The result shows the amount of total active/living cells, or how much microbial load or ‘life’ is in your sample. It is an easy and accurate method to assess the success of biocide treatments, and for routine microbial monitoring.

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

*Reagent Z is manufactured and shipped in bottles of freeze-dried powder and liquid buffer. The stated shelf life is for the freeze-dried form; store components in the refrigerator. Following rehydration, Reagent Z will be stable for 3 months when refrigerated and 6 months when frozen.

When you receive the Reagent Z and Buffer, you have a couple options. If the intent is to use the reagent immediately, you can refrigerate it in which it will remain stable for 3 months. If not, it is recommended that it be kept frozen and it will be stable for 6 months. Once you mix and the enzyme is in liquid form, refrigeration is the preferred method for storage.

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.

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 living microbial content in your sample. A drop in ATP can be directly attributable to the killing action of a biocide. Our LifeCheck ATP test kit is an easy, fast, and accurate microbial enumeration method, valuable in routine monitoring and supporting microbial management programs.

Culturing microbes is very difficult and will generally result in an underestimation of the total microbial load in a sample. Microbes are very picky about the exact conditions (temperature, salinity, nutrients) they need to grow. Culture media rarely satisfies these conditions, and leaves microbes undetected. Additionally, there are many microbial weeds that dominate in culture conditions but may not be representative of what is happening in the sample. When you compare a growth-based test to an ATP (or DNA) test (that does not require culturing), the ATP or DNA test will typically have a higher result because they are able to measure what a culture-based test cannot.

ME/ml	Bottle Turns
101	1
102	2
103	3
104	4
105	5
106	6

 

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 do not have a linear relationship to microbial issues, therefore there is no exact answer for contamination levels. This is simply a baseline to work from.

Culture media: take a sample and create a dilution series and then allow the bottles to incubate for a time ranging from at least 14 days (APB bottles) to 28 days (SRB bottles). The number of bottles that change color represents the log number of microbes present in your sample. IE. if you prepare 6 serial-dilution bottles and the first 3 change color, then you have 10³ microbes/mL in your sample.

BART (biological activity reaction test): 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 serial dilution and BART tests are growth-based methods, which has its drawbacks as indicated below.

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 an agar 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. The same is the case 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 effectively 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.

There is a high potential for false negatives as many microbes simply cannot grow in the culture media, even ones that are of interest (less than 1% of microbes are culturable even under perfect lab conditions). 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 implications.

Tests alternative to culture media 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 active/living 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/primers 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. OSP pairs a total microbes qPCR count with LifeCheck DNA 16S to provide this additional context.

These additional molecular methods do require a lab and a skilled technician to carry them out.

• LifeCheck DNA 16S Sequencing characterizes and provides insight into the entire microbial community present, and in what relative proportion. Once the microbial community makeup is determined, a trained microbiologist can determine the types of functions (harmful activities) the microbial community may be capable of.
• LifeCheck DNA qPCR is innovative genetic testing that allows the user to target and accurately quantify specific subgroups or types of microbes without the need for growth media.

The reason MIC is defined as “influenced” is because the bacteria don’t directly corrode the metal – 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.

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, their behavior is similar, and our control methods are employed in the same way.

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.

2K7 Features and Benefits:

• Broad spectrum, non-selective antimicrobial chemistry
• Non-ionic, highly compatible with other industrial chemicals
• Balanced efficacy (good balance between speed and length of protection)
• Available in multiple delivery mechanisms including dry add, solid biocide

Yes. 2K7 is non-ionic and therefore very compatible with most system components within the oil and gas industry. 2K7 has been successfully utilized in a variety of industrial settings for over three decades. 2K7 is a non-ionic highly compatible chemistry, widely used in oil and gas (fracturing and production), pulp and paper, and industrial water treatment.

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.

1. pg of ATP/ml: total mass of ATP in a sample. More ATP means more microbes
2. Colony Forming Units (CFU): Count the number of viable bacteria cells in a sample. Traditionally done on agar plates
3. Relative Light Units (RLU): photon (light beam) emitted when the luciferase enzyme contacts the ATP found in living cells
4. Bottle Turns: serial dilution log growth (Most Probable Number, MPN)

What is the comparable unit of measurement for all of the above?
ME/ml (microbial equivalents/ml) – number of microbial cells per unit volume or mass
ME provides a universal unit, directly comparable to the results of other common microbe quantification assays such as bug bottles (serial dilution log growth), CFU counts, microscopy counts and qPCR.

For example:
ME/ml = pg of ATP/ml x 1000 (The multiplication factor used is based on the amount of ATP found in an E. coli microbe, which has been determined to be comparable to the microbe species being evaluated in our industry.) ME/ml ~= CFU/ml

Microbial equivalents are a number of number of microbial cells per unit volume or mass – (ME/mL or ME/g). ME provides a universal unit directly comparable to the results of common microbe quantification assays such as bug bottles (serial dilution log growth), CFU counts, microscopy counts, ATP and qPCR. For more information, read comparing units of measurement – above.

Biocides are not “one size fits all”. A kill study is a laboratory experiment designed to quantify biocide performance for a given scenario. Also known as a biocide selection study, they attempt to simulate various treating conditions in order to determine the best fit biocide for a given fluid and application. Application environments, process components and fluid systems are mirrored to provide representative data and an understanding of the products behaviour in a specific application. To be the most representative, the exact water or fluid to be treated should be used in the study. Additional factors a well-designed kill study will consider include, biocide dose, application contact time, temperature, fluid chemistry and application compatibility.

Effective operations and longevity of asset life rely on proper microbial control programs. Like all pre-job performance tests, kill studies inform field decisions by providing insight into biocide product performance and providing a starting point for field optimization.

Biocide use includes not only understanding the objectives of control, but also what influences the performance of the various biocides. The outcome of a biocide selection study is laboratory validation of which biocide (and at what dosage) is likely to be the most effective, specific to your criteria. It takes the guessing out of the equation and drives cost efficiency focused on your pre-determined KPI’s.