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Updated: Aug 2, 2026
Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
Quantitative extraction of adenosine triphosphate from cultivable and host-grown microbes: calculation of adenosine
Abstract:
EXISTING DATA ON ADENOSINE TRIPHOSPHATE (ATP) POOLS IN MICROBES ARE DEFICIENT FOR TWO REASONS: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei, and Mycobacterium lepraemurium. Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time (E. coli, 3 min; M. phlei, 5 min; M. lepraemurium, 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.
Insights
This study introduces a novel method for accurately measuring adenosine triphosphate (ATP) in microbes. The optimized procedure ensures complete extraction and reliable quantification of microbial ATP pools.
Area of Science:
- Microbiology
- Biochemistry
Background:
- Existing methods for adenosine triphosphate (ATP) extraction from microbial samples are often incomplete.
- Extraction procedures can adversely affect standard ATP measurements and ATP released from bacterial cells, leading to inaccurate data.
- Previous methods failed to account for these extraction-induced artifacts.
Purpose of the Study:
- To develop and validate a new extraction method for accurately determining adenosine triphosphate (ATP) pools in microbial species.
- To overcome the limitations of existing ATP extraction techniques, including incomplete yields and procedural artifacts.
- To establish a reliable method for quantifying microbial ATP relevant to various biological studies.
Main Methods:
- A new extraction protocol was developed involving vortexing with chloroform, followed by heating at 98°C for specific durations based on microbial species (E. coli, M. phlei, M. lepraemurium).
- The method included a final vacuum drying step at 98°C for 1 minute.
- Traditional methods using perchloric acid and n-butanol were disqualified due to incomplete extraction and procedural complexities.
Main Results:
- The novel method successfully liberated 100% of the adenosine triphosphate (ATP) pools from all three tested bacterial species.
- The procedure demonstrated minimal impact on standard ATP, ensuring accurate baseline measurements.
- The extraction process avoided ionic imbalance and quenching of bioluminescence, crucial for sensitive ATP detection.
Conclusions:
- The developed method provides a comprehensive and accurate approach for microbial adenosine triphosphate (ATP) pool determination.
- This technique addresses critical deficiencies in existing methods, offering improved reliability for microbiological and biochemical research.
- The findings suggest potential applicability to plant and animal cells, broadening its utility in biological sample analysis.
