Detecting ATP bioluminescence using Amersham™ ImageQuant™ 800

Summary

Luciferase based assays use bioluminescence to detect ATP produced by viable cells. Cells are lysed to release ATP in solution before detection. Therefore, the first step for ATP measurement is selection and optimization of a lysis protocol. We compared detergent-based lysis to ultrasonication and detected the emitted light using Amersham™ ImageQuant™ 800 imaging system. The results of our analysis confirmed that different bacteria exhibited different lysis susceptibilities.

Introduction

Adenosine triphosphate (ATP) is a molecule at the core of living organisms, delivering energy to drive metabolic reactions, transport substances across membranes, and do mechanical work. Cells continuously synthesize and break down ATP to obtain energy. Most of the ATP in cells is produced by the enzyme ATP synthase, which is located in the membrane of mitochondria and chloroplasts.

A common way to detect ATP is to perform an assay using the enzyme luciferase. ATP is consumed and light is emitted having a wavelength of around 550 to 570 nm when luciferase catalyzes the oxidation of D-luciferin. In this article we used the Amersham™ ImageQuant™ 800 imaging system to detect emitted light from different bacterial samples to quantitate the amount of ATP within the cells.

When cells die, they stop synthesizing ATP and the existing ATP pool is quickly degraded. As a result, the amount of detected ATP has been widely accepted as a valid marker of the number of viable cells. Such measurements are frequently used to test hypothesis about cell growth, cell proliferation, and cytotoxicity. Furthermore, ATP assays are also used to determine the presence of bacterial contamination in water and food.

To measure intracellular ATP, it is necessary to lyse the cells. A plethora of different cell lysis methods have been developed for a variety of applications. Some of the most common physical disruption methods are:

• mechanical grinding
• shear-force disruption
• sound-wave sonication
• freeze-thaw cycles
• shaking the sample with beads

However, physical methods may lead to local heating and sub-fractions, and often require large volumes of sample. A popular alternative is to use milder solution-based cell lysis buffers. They often contain detergents to break the lipid cell barriers and solubilize cell components. Additional additives which are often included are salts and reducing agents. Depending on the application, various enzymes can be added, for example, lysozymes, DNases, RNases, and also protease inhibitors. We compared lysis with soundwave ultrasonication to lysis with a buffer for four different bacteria. We found the Amersham™ ImageQuant™ 800 imaging system to be well suited for detection of luciferase bioluminescence.

Results and Discussion

Cells were first centrifuged to form a pellet and the supernatant was analyzed. The results in Figures 1 and 2 show that the extracellular ATP levels were very low and difficult to detect. Thus, there was a need for cell lysis to analyze ATP from these microorganisms.

The four different bacteria samples were split into two fractions, one was treated with ultrasonication and one with lysis buffer. The released ATP in solution varied significantly depending on the bacteria and lysis method. E. coli was more efficiently lysed with the lysis buffer (Fig 2 and 3). However, Micrococcus and Bacillus were better lysed by ultrasonication (data not shown).

One plausible explanation is that E. coli is a gram-negative bacteria with a thin peptidoglycan layer in the cell wall and an outer membrane. They may be more susceptible to chemical lysis compared to gram-positive bacteria, which have a thicker peptidoglycan layer. Bacillus and Micrococcus are both gram positive bacteria and have a substantial cell wall, which in some cases may comprise as much as 50% of the cell mass. This could explain why the shear forces from sonication lysed the gram-positive bacteria more efficiently.

Fig 1. Plate analysis was performed using ImageQuant™ TL 10.2 software. The first step in the analysis was to make the grid and choose a suitable grid spot diameter (left). The 3D view is a magnified view of the three E. coli supernatant triplicate sample wells, which shows the low bioluminescence variation of the assay (right).

Fig 2. A luciferase assay on a 96 multi-well plate was used to detect ATP in bacteria using Amersham™ ImageQuant™ 800 to detect the emitted light. Images were captured with no emission filter. Bacteria samples were split into two equal fractions, one was treated with ultrasonication and one with lysis buffer. The measurements were performed in triplicates in each row. Micrococcus samples are indicated with red boxes and E. coli with blue boxes.

Fig 3. Detected ATP levels for the different fractions of E. coli (error bars are ± 3 standard deviations), showing the lysis buffer treatment led to the highest yield of released ATP for E. coli.

Conclusion

The results of our investigation highlight the importance of cell lysis optimization for each sample type as the different bacteria species exhibited different susceptibilities to lysis methods. It is recommended to evaluate a few different lysis protocols to select a robust lysis method. For example, lysozyme can be added to the lysis buffer to help break up the cell wall of gram-positive bacteria. The low variation of the triplicate samples shows that the ATP assay is a robust method and can be used to confidently measure ATP in cells using the Amersham™ ImageQuant™ 800 imaging system.

Learn more about how the Amersham™ ImageQuant™ 800 imaging system and ImageQuant TL image analysis software.

CY34376-21Aug23-AN