Differences in Bacterial Optical DensityMeasurements between Spectrophotometers Brian C. Matlock, Richard W. Beringer, David L. Ash, Andrew F. Page, Thermo Fisher Scientific, Wilmington, DE, USAMichael W. Allen, Thermo Fisher Scientific, Madison, WI, USA

Background

Optical density (OD) measurement of bacterial cultures is acommon technique used in microbiology. While researchershave relied on UV-Visible spectrophotometers to makethese measurements, the measurement is actually based onthe amount of light scattered by the culture rather than theamount of light absorbed. In their standard configuration,spectrophotometers are not optimized for light scatteringmeasurements; commonly resulting in differences in measuredabsorbance between instruments.

Introduction

The standard phases of bacterial culture growth (lag, log,stationary, and death) are well documented, with the logphase recognized as the point where bacteria divide asrapidly as possible.1 Using a spectrophotometer to meas-ure the optical density at 600 nm (OD600) of a bacterialculture to monitor bacterial growth has always been acentral technique in microbiology. Three of the more common applications where bacterial OD600 is used arethe following: (a) determination and standardization of theoptimal time to induce a culture during bacterial proteinexpression protocols, (b) determination and standardizationof the inoculum concentration for minimum inhibitoryconcentration (MIC) experiments, and (c) determination ofthe optimal time at which to harvest and prepare competentcells. Researchers continue to rely on absorbance spectrophotometers to make these OD measurements.Optical density, however, is not a measure of absorbance,but rather a measure of the light scattered by the bacterialsuspension which manifests itself as absorbance. The effectthat the optical configuration of a spectrophotometer has onoptical density measurements has been well documented.2-4

Instruments with different optical configurations willmeasure different optical densities for the same bacterialsuspension. Differences in the optical configuration of thespectrophotometer make the largest contribution to the

observed differences. Forward optical systems employmonochromatic light for the measurement of absorbancewhere reverse optical systems utilize polychromatic radiationthat is discriminated into individual wavelengths after it ispassed through the sample. Some components of the forwardoptical systems contribute to a difference in measured OD values: (a) the distance between sample and detector,(b) the size and focal length of any collector lens used, and(c) the area and sensitivity of the detector.5 Despite currentknowledge that different optical configurations will givedifferent OD values, researchers continue to raise concernsabout the differences in OD values seen among differentspectrophotometers. In this study, we analyzed OD valuesobtained from several spectrophotometers possessing variousoptical configurations. E. coli JM109 was grown in batchculture and the OD of the culture was monitored for 9.5 hours. Our measurements demonstrate the need todilute cultures before measurement and show how toapply a conversion factor to the OD data in order to normalize differences in OD values due to the opticalgeometries found in different spectrophotometers.

Key Words

• Bacterial Growth

• E. coli

• OD600

• Optical Density

• UV-VisibleSpectrophotometers

Technical Note: 52236

Materials and Methods

Strain

E. coli JM109-endA1, recA1, gyrA96, thi, hsdR17 (rk–, mk

+), relA1, supE44, �(lac-proAB), [F′ traD36, proAB,laqIqZ�M15] (Promega, L2001)

Spectrophotometers

Thermo Scientific UV-Visible spectrophotometers used aredescribed on Table 1.

Growth Curve

A 50 mL overnight E. coli JM109 culture was grown in a250 mL baffled flask (16 hr, 250 rpm, 37 °C) in LuriaBertani (LB) Broth. A batch culture was prepared bytransferring 12 mL of the overnight culture in 600 mL ofpre-warmed (37 °C) LB media in a 2 L baffled flask. Thebatch culture was grown for a total of 9.5 hours.

Culture Sampling

All spectrophotometers were blanked using LB broth.Every 30 minutes, a 5 mL aliquot was sampled from thebatch culture. A 3 mL aliquot of undiluted culture wastransferred to a 10 mm cuvette, and the optical density at600 nm was measured on all the instruments listed abovein Table 1. A second 10 mm cuvette was prepared with analiquot of the batch culture diluted in LB medium to anOD600 of approximately 0.5. This second OD measurementwas measured to ensure that the optical density of the culture remained within the dynamic OD range of thespectrophotometers.

Viable Counts

At each 30 minute interval, an aliquot of the sample wasalso used to perform serial dilutions. Dilutions were platedon LB Agar plates and incubated at 37 °C overnight.Colonies were counted to determine bacterial cell count(CFU/mL) at each time point.

McFarland Standards

OD600 measurements of aliquots from four McFarlandstandards (Remel, R20421, 1.0 – 4.0) were measured oneach instrument (Table 1) by using 1 cm pathlength cuvettes.

Figure 2: Differences between forward and reverse optical systems.

(A) In a forward optical system monochromatic light passes through the sample and then onto a detector.

(B) In a reverse optical system, polychromatic light passes through the sample, discriminated into individual wavelengths and measured on an array detector.

Figure 1: Light scattering in spectrophotometry.

(A) In a non-scattering sample, the attenuation in light transmission betweenthe light source and the detector is caused by the absorbance of light bythe sample.

(B) In a scattering sample, (i.e., a bacterial suspension), the light reaching the detector is further reduced by the scattering of light. This decrease in light reaching the detector creates the illusion of an increase in sample absorbance.

Results

OD600 data collected was from the undiluted and diluted cultures and representative growth curves are shown inFigure 3. OD600 values from diluted solutions were multiplied by the dilution factor and compared to undilutedsamples. Divergence of the two plots illustrates that differentoptical configurations will have different dynamic rangeswith respect to optical density measurements. When thecorrected OD values for each spectrophotometer and thecell counts were compared, we found that instruments withsimilar optical systems produced similar OD curves overtime (Figure 4). The BioMate™3S, which has a forwardoptical system but a dual-beam optical geometry, shows asystematically lower OD600 measurement than indicatedby the plate counting method, most likely due to itsunique optical geometry. The McFarland data shows asimilar trend as was observed with the E. coli growthcurves; similar optical systems grouped together (Figure 5).

Figure 3: Comparison of growth curves of E. coli JM109 defined by measuringOD600 of diluted or undiluted bacterial samples. OD600 measurements wereperformed on the Evolution 260 Bio and the Evolution Array instruments. OD measurements were carried out every 30 minutes for 9.5 hours. Blue linesrepresent the OD600 from diluted culture samples. Samples were diluted so they were within the dynamic range of the optical system. Green linesrepresent the OD600 of undiluted culture samples.

Figure 4: Growth curves of E. coli JM109 obtained from corrected OD values.The OD600 was measured on all instruments every 30 minutes for 9.5 hours.The OD600 data was corrected by the dilution factor used for OD measurement.Samples were diluted so they were within the linear range of the opticalsystem. The corrected OD for each spectrophotometer was then plottedalongside the viable cell count data.

Figure 5: The OD600 of various McFarland standards measured on spectrophotometers listed in Table 1

Instrument Optical System Optical Geometry Light Source(s) Detector

Thermo Scientific Evolution Array Reverse Optic Deuterium and Tungsten Lamps Photodiode Array

Thermo Scientific NanoDrop 2000c Reverse Optic Xenon Flashlamp CMOS Array

Thermo Scientific SPECTRONIC 200 Reverse Optic Tungsten Lamp CCD Array

Thermo Scientific Evolution 260 Bio Forward Optic Double Beam Xenon Flashlamp Silicon Photodiodes

Thermo Scientific Evolution 300 Forward Optic Double Beam Xenon Flashlamp Silicon Photodiodes

Thermo Scientific BioMate 3S Forward Optic Dual Beam Xenon Flashlamp Silicon Photodiodes

Table 1: Thermo Scientific spectrophotometers

Conversion Between Spectrophotometers

The variation in optical density observed between twoinstruments can make standardization of a protocol difficult.This is especially true when one laboratory tries to replicate data from another lab, but uses a different spectrophotometer, or when an aging spectrophotometer is replaced in the same lab.

Figure 6 shows the OD of a culture measured on bothan Evolution™ 260 Bio and a BioMate 3S. The OD ratiobetween the two instruments was calculated at each timepoint. The OD600 ratio from each time point was deter-mined and the average ratio was calculated and used as amultiplication factor. For the BioMate 3S, the calculatedconversion factor was 1.46. Application of this correctionfactor to the OD data normalizes the data and facilitatescomparison between both instruments. To better facilitateagreement among spectrophotometers, a correction factoris included in the local control software of the BioMate 3Sspectrophotometer.

Conclusion

It is critical to determine the optical density performanceand dynamic range for your spectrophotometer when calculating OD measurements. For the most dynamicinformation about the growth curve it is important todilute the cell suspension.

OD measurements are highly dependent upon theoptical system and geometry. Spectrophotometers with different optical systems and configurations will read different OD values for the same suspension. For example,there is substantial divergence between the reverse opticalsystem Evolution Array™ and the forward optical system,dual-beam BioMate 3S.

Finally, we present how to calculate a conversion factor that can be used to normalize the OD data so that appropriate comparisons can be made between spectrophotometers. It is important to note that this conversion factor is specific to a particular organismbecause the size and shape of the particle will affect theconversion factor.

References1. Daniel R. Caldwell, Microbial Physiology and Metabolism (Wm. C.

Brown Publishers, 1995), 55-59

2. Arthur L. Koch, “Turbidity Measurements of Bacterial Cultures in SomeAvailable Commercial Instruments,” Analytical Biochemistry 38 (1970): 252-59

3. Arthur L. Koch, “Theory of the Angular Dependence of Light Scattered byBacteria and Similar-sized Biological Objects,” Journal of TheoreticalBiology 18 (1968): 133-56

4. J.V. Lawrence and S. Maier, “Correction for the Inherent Error in Optical Density Readings,” Applied and Environmental MicrobiologyFeb. (1977): 482-84

5. Gordon Bain, “Measuring Turbid Samples and Turbidity with UV-VisibleSpectrophotometers,” Thermo Scientific Technical Note 51811

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Figure 6: Application of a conversion factor to compare OD600 data from two different spectrophotometers. Green = original BioMate 3S data; Red = BioMate 3S data × factor of 1.46.

Figure 7: Equation Calculationfor conversion factor betweenspectrophotometers. For themost accurate conversion factortwo things are important; (1) AllOD measurements used to calculate these values are from the corrected OD data (Figure 3), (2) take the average multiple conversion factor that arecalculated across a range of various OD measurements.