Comparative Study Highlighting the Application of Absorbance 96 for OD600 Measurements

Key highlights

• OD values obtained by Absorbance 96 were nearly identical to those obtained by using another commercially available plate reader. 
• Care must be taken when comparing different microplate readers from different manufacturers, as their differences in optical design can impact OD readout. 
• The compact design of Absorbance 96 offers benefits, such as the ability to perform on-site OD measurements — enhancing lab efficiency and productivity.

Introduction 

Bacterial growth measurements are a crucial aspect of many research fields, including fermentation, antibiotic efficacy, synthetic biology, as well as the food industry. Estimation of bacterial growth provides insight into the dynamics of the bacterial population, its growth rate and activity, and the effects of environmental conditions on these processes. By understanding the growth rate, it is possible to determine the optimal conditions for bacterial proliferation and maximize the yield of desired products such as metabolites, and recombinant proteins in biotechnological applications. Additionally, examining the factors underlying factors influencing bacterial growth facilitates the development of targeted strategies for the control and inhibition of bacterial proliferation, particularly in the case of pathogenic bacteria. 

There are various methods to quantify bacterial growth. Optical density (OD) measurement is the most common and easy method to measure bacterial growth. OD measurement is based on light scattering when a beam of light (usually at 600 nm) is passed through the bacterial sample — more bacteria in the sample will scatter more light. Thus, the method is applied to estimate the number of bacteria in a sample, since the measured value is directly proportional to the amount of bacterial biomass. As the number of bacteria increases, the OD600 value will also increase (1,2). 

This application note shows how the Absorbance 96 plate reader is useful for determining OD600 measurements. In collaboration with HM Munich University of Applied Sciences, a comparative analysis of Absorbance 96 with a different spectrophotometer and a different microplate reader was performed to monitor the bacterial growth of a ureolytic bacterium Sporosarcina pasteurii, well-known for its unique capability of microbially induced calcite precipitation (MICP) required in constructional engineering and material applications (3). The OD readouts obtained by Absorbance 96 are comparable with the readouts obtained by other commercially available plate readers, confirming the reliability and efficiency of Absorbance 96 for OD measurements. Additionally, the small footprint of Absorbance 96 is an extra advantage compared to other traditional, large-size plate readers — offering flexibility and easy workflow in any laboratory or outside field applications. 

Results

Absorbance 96 and SpectraMax iD3 plate readers showed identical OD values

Bacterial growth can be quantified by accessing the optical densities of the samples. Therefore, absorbance measurements using Absorbance 96 and SpectraMax iD3 microplate readers were conducted to compare their measurement accuracies. Triplicate OD measurements of the dilution series of Sporosarcina pasteurii in a 96-well microplate were performed at 605 nm, first with SpectraMax iD3, and subsequently, the same microplate was measured with Absorbance 96. The obtained OD605 values from both devices were practically identical and showed no significant difference in measurements (T-test: p-value > 0.005, (0.744)). This could be explained by the similar optical design of both the Absorbance96 and SpectraMax iD3. Thus, these results highlight that OD measurements performed by Absorbance 96 are highly comparable to other traditional plate readers. 

Photometer measurements recorded higher OD values

Measuring the optical density of the bacterial samples at 605 nm with BioMate 3 photometer showed slightly higher OD605 values compared to those values obtained by the plate readers (SpectraMax iD3 and Absorbance 96) (Fig. 1). This difference in the absolute value of OD605 measured by the photometer can be explained by how the light beam is delivered into the samples and the optical design of the devices. The light beam passes horizontally through the cuvettes in photometers, and the path length is usually standardized to 1 cm. However, in plate readers, the light beam passes vertically through the wells, and this orientation creates variables that need to be accounted for. In this case, the volume in the wells determines the path length. The accuracy and consistency of pipetting may also affect the OD readouts (4).

Moreover, this measurement is based on light scattered by the microbial organism rather than the amount of light absorbed, therefore, the Beer-Lambert law, which establishes a linear relationship (A=εbC; where A is absorbance, b is the path length, ε is molar absorbance, C is the bacterial concentration) between absorbance and concentration, is theoretically not applicable to turbidity measurements. However, Beer-Lambert law can still be applied to measure bacterial concentration within the consideration of the low-density microbial culture (2). 

Thus, two different OD values will be obtained by measuring the OD of identical samples in cuvette based- photometers and plate readers due to the difference in the path length. This explains the differences in OD values that were observed while using different devices as shown in Fig 1. 

Discussion

In this application note, we conducted a study of OD measurements obtained with the Absorbance 96 plate reader compared with two other commercially available instruments: the BioMate 3 photometer and SpectraMax iD3 plate reader. BioMate 3 showed relatively higher OD605 values compared to both plate readers, which can be explained due to the difference in the path length and the optical design of the instruments. However, the OD605 results obtained by Absorbance 96 and SpectraMax iD3 (plate readers) were practically identical, suggesting that Absorbance 96 can effectively measure bacterial growth compared to any other plate reader. These observations also suggest that users need to be careful when comparing OD600 readings between different spectrophotometry instruments because the optical design of the instruments impacts the resulting OD600 values. 

Plate readers' optical design influences OD measurements     

When comparing two different plate readers from different manufacturers, one cannot expect to get the same OD600 readouts, even when using the same sample. The determining factors here are the size of the aperture and the distance between the sample and the detector. If the size of the aperture is bigger, it will collect slightly more scattered photons than a smaller one, And, depending on the position of the detector with respect to the sample, if the detector is far away from the sample, it collects fewer scattered photons, thus impacting the OD600 value. Since every manufacturer has a different inbuilt optical design, the above-mentioned parameters are never the same in different plate readers. Thus, photometers and plate readers must be cross-calibrated to compare OD600 readings as absolute values (2). 

Absorbance 96 offers a simple workflow solution for bacterial growth measurement    

The OD605 results obtained from Absorbance 96 and SpectraMax iD3 plate reader were almost identical. Nevertheless, considering the workflow for OD600 measurements, Absorbance 96 offers several advantages over other commercially available devices in the market. First, the compact size of Absorbance 96 facilitates easy mobility, allowing for on-site OD600 measurement directly at the workstation (e.g. fermenter) without the need for sample transportation to other locations housing plate readers. Additionally, software for operating Absorbance 96 and further data analysis is very user-friendly, allowing quick OD600 readouts and insights. 

Materials and experimental outline  

To evaluate the effectiveness of Absorbance 96 for measuring bacterial growth, OD measurements for Sporosarcina pasteurii were conducted in different spectrophotometry devices and the readouts obtained were compared. 

First, bacterial culture of Sporosarcina pasteurii (obtained from German Collection of Microorganisms and Cell Cultures; DSMZ) was prepared by cultivating it overnight in 20 mL of Caso medium (Merck 105458) supplemented with 4 mL of urea (20g/L) at 30°C and 200 rpm. On the following day, the bacterial culture was diluted (1:10), and a series of dilutions (0%, 10%, 25%, 40%, 55%, 70%, 85%, 100%) of 10 mL were prepared in a Caso medium. For comparison, first OD was measured with the BioMate 3 photometer (Thermo Fisher Scientific) at 605 nm (OD605) in triplicates by incubating 1 mL of the bacterial culture sample in semi-micro cuvettes (LabSolute). Here, the blank was determined by setting it to 0 %. Next, a 96-well microplate was incubated with the different dilution series samples (above-mentioned) and the OD was measured by SpectraMax iD3 (Molecular Devices) microplate reader without a lid at 605 nm in triplicates. Then, the same 96-well microplate was used (without shaking) to measure OD with Absorbance 96.  
 

References 

1. Mandelstam J, McQuillen K, Dawes, IW. Biochemistry of Bacterial Growth.1982, 449.
2. Stevenson K, McVey AF, Clark IBN, Swain P. S, Pilizota, T. General Calibration of Microbial Growth in Microplate Readers. Sci. Reports 2016 61 2016, 6 (1), 1–7.
3. Ma L, Pang AP, Luo Y, Lu X, Lin F. Beneficial factors for biomineralization by ureolytic bacterium Sporosarcina pasteurii. Microb Cell Fact. 2020 Jan 23;19(1).
4. Beer. Bestimmung der absorption des rothen lichts in farbigen flüssigkeiten. Ann. Phys. 1852, 162 (5), 78–88.