Using Infinite® 200 PRO Multimode Microplate Reader for Determination of Microbial Cell Viability

Introduction

Microbial cell viability is determined by using a luminescence-based assay called BacTiter-Glo Microbial Cell Viability Assay. This assay uses ATP quantification as an indicator of viable and metabolically active cells (1).

The chemistry of the assay is based on the properties of a thermostable luciferase (Ultra-Glo Recombinant Luciferase), shown in Figure 1, and a proprietary formulation to extract ATP from bacteria.

In the assay protocol, the BacTiter-Glo reagent is added directly to the sample and the luminescence is subsequently measured. Removal of culture medium, cell washing, and multiple pipetting steps are not needed, as shown in Figure 2.

The resultant luminescent signal is proportional to the quantity of ATP, and therefore indicates the number of viable cells present in the sample. Thanks to the ‘glow-type’ nature of the assay, the signal can be measured over a period of 30 minutes, dependent on the growth medium and the type of bacteria.

Figure 1. The luciferase reaction (1, copied with the permission of Promega Corp.).

Figure 2. Schematic of the BacTiter-Glo Microbial Cell Viability Assay protocol (1, copied with the permission of Promega Corp.).

The Infinite F200 PRO filter-based multimode reader was used to test the BacTiter-Glo Microbial Cell Viability Assay, by employing the highly sensitive luminescence module of the instrument.

Materials and methods

The following instruments were used:

  • Infinite F200 PRO filter-based multimode reader (Tecan, Austria)
  • BacTiter-Glo™ Microbial Cell Viability Assay kit (Promega, USA)
  • 96-well, white polystyrol microplate (Greiner®, Germany)

According to the assay instructions, the BacTiter-Glo Microbial Cell Viability Assay reagents were prepared (1). Again, according to the assay instructions, a simple ATP dilution curve was prepared to validate the assay (1), and measured on the Infinite F200 PRO reader using the instrument settings (Table 1).

In addition, the assay was employed to detect the bacterial burden of commonplace items and publicly accessible locations, including:

  • Laboratory floor
  • Computer mouse
  • Tap water
  • Toilet water
  • Laminar flow hood bench (for cell culture)
  • Dish washing sponge

Using sterile cotton buds, samples were collected from the laboratory floor, computer mouse, and the laminar flow hood bench. Then, the buds were transferred to an Eppendorf tube containing 500 μl ATP-free H2O.

Approximately 500 μl of samples were taken directly from tap water, toilet water, and the dish washing sponge. The manufacturers’ instructions were followed for preparing the assay, i.e. 15 minutes of incubation time for both the samples and the ATP dilution series.

Table 1. Infinite 200 PRO measurement parameters and instrument settings for the BacTiter-Glo Microbial Cell Viability Assay

Measurement parameter

Instrument settings

Plate

GRE384sw.pdfx

Mode

Luminescence

Attenuation

Automatic

Integration time

1,000 ms

Settle time

0 ms

 

Results and discussion

Figure 3 depicts the ATP dilution curve determined on the Infinite F200 PRO, showing perfect linearity over five orders of magnitude.

Figure 3. ATP dilution curve.

The bacterial burden of a few commonplace items and publicly accessible locations are shown in Figure 4. Expectedly, the toilet water, dish washing sponge, laboratory floor, and computer mouse showed a substantial level of bacterial contamination, while the tap water and the cell culture lamina flow hood bench remained at control level.

Figure 4. Bacterial burden determined using the BacTiter-Glo Microbial Cell Viability Assay and the Infinite F200 PRO reader.

Conclusion

This study has shown that Tecan’s Infinite F200 PRO filter-based multimode microplate reader is compatible with Promega’s BacTiter-Glo Microbial Cell Viability Assay. This compatibility enables determination of the bacterial burden of various materials. Perfect linearity is shown by the determined ATP dilution curve over a dynamic range of more than five orders of magnitude.

Literature

1) Technical Bulletin: BacTiter-Glo Microbial Cell Viability Assay. Instructions for use of products G8230, G8231, G8232 and G8233, part# TB337, Promega.

About Tecan

Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: May 23, 2017 at 6:06 AM

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