ATP Testing: Principle, Procedure, Applications

ATP is an abbreviation of adenosine triphosphate, an organic compound produced that is commonly known as the cell’s energy currency. It provides energy to different cellular processes like metabolism and chemical synthesis. ATP is measured because it serves as an excellent marker for detecting the microorganisms in the skin of humans and other inanimate objects as bacteria and yeasts produce ATP during different biological processes. 

You have spent an hour cleaning your workspace before starting work. Are you sure the workspace is spotless? Traditional methods of monitoring the surface’s cleanliness are effective but time-consuming and tedious. ATP testing helps to detect the ATP produced by live microorganisms like bacteria from various surfaces and water, which helps assess the hygiene in the food industry and test the water quality quickly. But ATP testing does not help detect viruses as they do not produce ATP

Requirement for ATP Testing (ATP Testing Kit)

The ATP testing requires the following materials for carrying out the test:

  • Swab: A sterile swab helps collect samples from the skin and inanimate objects. The swab should be such that it will be easy to bend and break off. Also, the swab used is unique due to the presence of luciferin/luciferase enzyme is already present in it. Usually, a biodegradable swab like ultrasnap is used.
  • Syringe: A syringe of 10 ml is preferred for testing water and food samples.
  • Filter: It is attached to the syringe for filtering the water sample.
  • Tube: A tube that is used to place the water sample. GL bicontrol’s lumitube is commonly used.
  • Luminometer: It is the equipment that measures the intensity of light.

Principle of ATP Testing

Bioluminescence is the principle of ATP testing. It means detecting illuminating light that is produced by the oxidative decarboxylation of luciferin in the presence of ATP, magnesium ions, and luciferase enzyme. 

The detailed reaction is as follows:

Luciferin+ O2+ ATP → oxyluciferin+ AMP+ light; in the presence of Mg2+ and luciferase. Here, AMP is adenosine monophosphate.

The luciferase enzyme catalyzes the change of luciferin to oxyluciferin, resulting in the production of light. Thus emitted light has an intensity proportional to the amount of ATP present in the sample. It is because the increased ATP levels degrade more luciferin. The optimum pH of the test should be 7.8-7.9 and the optimum temperature 25℃. Luminometer is used to detect light. The wavelength is 562 nm, and the light unit is RLU (relative light unit). 

The total ATP (tATP) is the sum of cellular ATP (cATP) and dissolved ATP (dATP). cATP occurs in the living cells, whereas dATP arises from decomposed cells. Thus, the cATP analysis in water and wastewater samples helps us know that the concentration of the bacteria (anaerobic and aerobic). It is assumed that 1 pg of ATP equals 1000 bacterial cells for estimating the number of bacterial cells.

pg= picogram

ATP testing
Full ATP testing Kit
Image source:

Procedure of ATP Testing

The procedure depends on the type of sample and the instruments used.

Here, three ways of performing ATP testing are explained.

ATP Testing for Water Sample

Source: GL Biocontrol

The procedure for ATP testing for water samples using GLBiocontrol’s ATP testing kit is as follows:

  • First, turn the luminometer on for calibration and ensure the reagents are at room temperature before analysis.
  • Then, place a lumitube in the tube holder. 
  • After that, remove the plunger from the syringe. Do not touch the inner part of the plunger and tip of the syringe. 
  • Then, fit a filter in the tip of the syringe. Please do not touch the filter and use it directly from the plastic container it is packed in.
  • After that, fill precisely 10 ml of water from the sample container in the syringe. Put the plunger back.  
  • Then, filter out the water from the syringe in the sample container and place the filter-fitted syringe on a clean surface.
  • Then, add 3-4 drops of extractant to the plastic container of the filter. With the help of the filter-fitted syringe, plunge out the liquid from the plastic container.
  • After that, pour the plunged solution into the lumitube until white foamy liquid appears.
  • Then, clip the lumitube into the end of the tube holder and place the tube holder in the luminometer. 
  • Finally, start the luminometer and generate the results in about 15 seconds. 

ATP Testing for Inanimate Objects

The procedure below is for ATP monitoring by Hygiena. 

  • Firstly, please turn on the Hygiena luminometer for at least a minute to calibrate it. 
  • Then, select the area you want to check. And bring out the ultrasnap swab from the tube.
  • After that, swab the area chosen with even coverage. The swab is bendable, so apply enough pressure to bend it. Be careful not to touch the swab’s shaft and rotate it with every swabbing. 
  • Then, place the swab back into the tube. Break the bulb end of the swab with the index finger and thumb.
  • Then, squeeze the remaining liquid from the bulb into the tube.
  • After that, place the tube in the tube holder section of the luminometer. 
  • Finally, press start once you are sure the machine is ready.
  • It takes about 10-15 seconds to show the results.
Source: Hygiena

ATP Testing in Human Hands

The procedure for ATP monitoring in human hands is the same as testing in inanimate objects. Care must be given, ATP testing in hands is done twice, before and after washing, to reduce contamination, and all parts of the hands are covered while swabbing.

Standard Value of ATP Testing

Once you perform the test and obtain the result, you must be wondering what the result means. So, the RLU displayed in the luminometer is now compared with the standard value of the test. The standard value is different based on the industry performing it or the kit you use. Anything below 10 RLU is considered clean in the food industry, whereas anything above 10 contains pathogenic biofilms. Likewise, the standard value in water ATP testing varies based on the region, state, or country. 

A chart by Zbioscience shows the comparison between ATP readings and the probability of pathogenic biofilms:

ATP reading (RLU)Probability of pathogenic bacteria

Similarly, another way of setting the target/standard for your industry/laboratory/company is by conducting thorough research by performing the tests numerous times. 

For example, research by Meritech shows the comparison of pass/fail limit and percentage reduction in the microbes in the hands of their workers. On this basis, they have determined the limit of the pass or fail for working in the laboratory.

WorkerPre-Wash RLUPost-Wash RLUPercent reductionPass/Fail 100 RLU limit
112027Invalid testInvalid

Application of ATP Testing

The use of ATP testing is in various fields for monitoring hygiene as well as the quality of the products generated.

ATP Testing in the Food Industry 

In the food industry, ATP testing helps detect the quality of food products. It also helps determine the production area’s hygiene status and verify the cleaning measures used by food technicians. 

ATP Testing in Testing Water Quality

  • Since water can potentially cause diseases like cholera, diarrhea, typhoid fever, and many more diseases. So, detecting ATP levels helps control the contamination of water at low levels.
  • Likewise, the pipelines distributing water may corrupt due to biofilm formation. Thus, the ATP test helps prevent biofilm formation in the water distribution system.
  • Similarly, the industries use rivers and lakes as the source of water which contains heavy growth of microorganisms. The water is treated with various chemicals in order to prevent biofilm from corrupting the machines and equipment of the industry. That is why ATP testing helps to monitor the effectiveness of those chemicals.
  • ATP Testing also helps detect Legionella in water cooling tanks. Legionella is responsible for transferring severe pneumonia or Legionnaire’s disease.  

ATP Testing for Cleanliness

As the test detects ATP produced by microorganisms, it helps monitor the sanitary condition of different health facilities, microbiology laboratories, quality testing laboratories, etc. It also helps test the efficacy of hygienic/sanitation equipment and chemicals used. 

References and further reading

Ashma Shrestha

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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