Bacterial spores have a fascinating ability to endure extremely harsh environments¹. An example of this is the isolation of viable spores from plant samples dating back to 1640².Some spore-formers, e.g. Bacillus anthraxis, are notorious pathogens, but nonetheless, non-pathogenic spore-formers have received growing interest in live biotherapeutic products or as ingredient in probiotic foods and supplements. This interest is based on the fact, that – compared to vegetative cells – bacterial spores show superior survival through the acidic stomach so they can colonize the intestines and confer health protection to the host³. Apart from probiotics and live biotherapeutics, benign Bacillus spores are also formulated in cleaning products to suppress bacterial pathogens like Staphylococci and Streptococci on surfaces⁴. Last, but not least, spore-forming bacteria are finding applications within agribiotech to aid in plant growth promotion, disease resistance, quality improvements and abiotic stress⁵.

Two Firmicutes genera are the most exploited organisms for spore-production, namely the aerobic Bacillus species and the anaerobic Clostridia species. The present application note focuses primarily on Bacillus endospores. The sporulation process has been detailed elsewhere (for an excellent illustration, see McKenney et. al, 2012¹). Bacillus initially follows a vegetative growth cycle, but deprivation of nutrients or environmental stressors can trigger the sporulation cycle. Through an asymmetric cell division, the initial sporangium forms and the endospore starts to appear as the smaller part of the cell division, the forespore. Later in the sporulation process, the endospore is engulfed within the mother cell and will only be released upon mother cell lysis. When nutrient conditions are again favorable, the spore can enter the vegetative cycle¹. Thus, the endospore provides a convenient, well-protected “escape pod” to resist environmental stressors.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Two overall methodologies exist to enumerate bacterial spores: Cultivation or particle counting by e.g microscopic counts, flow cytometry and particle analyzers like the Coulter counter⁶.

Plate counts: The gold standard is cultivation by spread-plating, typically following a two-step protocol, where samples are plated before and after heat treatment^6,7. Before heat (BH) plate counts will show the total number of viable bacteria, i.e. both spores and vegetative cells, while the after heat (AH) result will only reflect the concentration of heat-stable endospores. While this is a simple and inexpensive method, substantial manual efforts are needed and the turnaround time can be several days. This is mainly due to the long germination time for all spores to “hatch”. Also, there are pitfalls that can lead to underestimation, for example endospores tend to agglomerate, which can lead to a ten-fold underestimation, and in addition, the early-germinating mucoid colonies of e.g. Bacillus can often hide late-sporulating colonies. The hydrophobic spores are also known to adhere to the typical glass or polymer Drigalsky spreaders used for distributing the sample on the agar⁶. Inter-operator variation is an additional well-known shortcoming of the traditional plate counts⁸.

Microscopy: The refractive properties of spores make them easy to distinguish from vegetative cells using phase contrast microscopy. Nonetheless, the small size of spores makes them challenging and tedious to enumerate in counting chambers, and the analysis is operator-dependent. The main
shortcomings with this technique are low throughput and low precision making it unfit for real-time monitoring of sporulation processes⁹.

Fluorescence flow cytometry (FFC): Compared to microscopy, FFC offers higher throughput and much higher precision. FFC is therefore regarded as a promising tool for real-time monitoring of sporulation processes and enumeration of spores in final products ^8–11. Despite their advantage FFC
instruments are expensive and extensive training is needed to achieve accurate results. In addition, FFCs are bulky and require tidy working environments for staining processes, sample workup and analysis making them unfit for monitoring sporulation processes close to the action.

Particle analyzers: Instruments like the Multisizer have excellent precision and can be exploited to determine the concentration of spores in highly purified spore preparations⁶. On the other hand, it is not straightforward to perform real-time monitoring of active sporulation processes, as it is difficult
to distinguish endospores from other cellular debris with similar size. Finally, size-based particle analyzers will likely not reveal endospores contained within mother cells with a size similar to that of vegetative bacteria.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

As highlighted above, there is a need for a precise, user-friendly, and operator-independent method for characterization of sporulation processes. We propose BactoBox as a robust, label-free tool for simultaneous monitoring of endospores and vegetative cells. The rationale for this is that the
electrical properties of endospores and vegetative bacteria are very different. The G⁺ vegetative cell of Bacillus has a single, isolating lipid membrane and a conductive cytoplasm. On the other hand, the spores have two sets of isolating forespore lipid membranes and relatively little conductive core
water content¹. The measurement principle in BactoBox is based on impedance flow cytometry (for more information, see Bertelsen et al., 20201²). Lipid membranes pose a significant hindrance (impedance) to current flow, and with the presence of two sets of lipid membranes, endospores are
expected to be less conductive than their vegetative counterparts. Also, the differences in size should result in smaller signal amplitudes for endospores than for vegetative bacteria when flowing through the electric fields in the BactoBox flow cell.

We therefore set out to exploit BactoBox for investigation of fermentation processes for two spore-producing bacteria, namely Bacillus thuringiensis (DSM 6108) and Bacillus subtilis subsp. spizizenii Bundesgesundheitsamt (BGA, DSM 618). B. thuringiensis displays a remarkable production of protein crystals (delta toxins) with a potent insecticidal effect on insect larvae, and in fact B. thurigiensis spores is the most commonly used biopesticide worldwide¹³. To release these toxins, the mother cells of B. thuringiensis must lyse efficiently, leading to a concomitant release of endospores^13,14. B. subtilis, on the other hand does not form crystal toxins and therefore the endospore-containing mother cell stage is more long-lived and it can be necessary to exploit various treatments to release the endospores, e.g. heat shock, surfactants, organic solvents, enzymes and ultrasound^15–17.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

The cornerstone for the study was to initially hone in on the optimal BactoBox settings for Bacillus endospores using a commercial purified preparation of endospores. Subsequently the goal was to perform real-time monitoring of sporulating Bacillus cultures. In brief, shake flasks were inoculated with a colony from an agar plate and samples were drawn during the early exponential phase and the later sporulation processes. At each time point, 5 mL sample was transferred to a 15 mL vial and homogenized using bead beating with 3 mm glass beads (Bead Genie, speed 4800, 1 min). All samples were diluted in a diluent of ~1850 µS/cm to provide adequate parameters for detection of intact cells and to fit the linear range of BactoBox. After that, samples were measured on a BactoBox in one-plicates. Samples were also inspected with phase contrast and fluorescence microscopy to achieve a qualitative assessment of the transition from vegetative cells to endospores. SYTO-9 was used as a total stain at a final concentration of 10x. TOTO-3 was likewise used at a 10x concentration. Staining was performed in the dark for 15-30 min.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

The electrical properties of endospores are expected to differ from that of normal, vegetative bacteria. Firstly, they are wrapped in two sets of poorly conductive lipid bilayers, and secondly the low water content in the core deviates from the normal highly conductive cytoplasm of vegetative bacteria.

To find the best BactoBox settings for endospores, BactoBox measurements were performed on commercially available purified spores from B. subtilis BGA. The suspension was vortexed briefly and diluted 1:100 in standard BactoBox diluent prior to BactoBox measurement. Two populations were observed in the phase shift diagram for untreated purified spores (upper histogram, black curve). The phase shift is a concept from electrical engineering which is best explained as a “delay” of the electrical signal as it passes through objects, e.g. bacteria and other particles. When analyzed with BactoBox, objects with a phase shift of ~1 rad are of an entirely non-conductive nature. The 1.1 rad population is therefore very poor at propagating electricity, while the distribution at ~1.7 rad is slightly more conductive, but still almost exclusively outside the more conductive region of normal intact vegetative cells (white region from 1.83 to 3.14). The amplitude distribution (lower histogram) is mainly reflecting the size of the objects. Two populations are observed with some overlap. The endospores are known to be small (0.5-1.5 µm), ellipsoidal or spherical, and they are therefore expected to belong to the distribution with the lowest amplitudes ranging from -74 to approx. -60.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Purified BGA spores reveal two populations with different electrical properties: First population is centered at ~1.05 rad in the phase shift histogram, while the second population is centered at ~1.7 rad. The x-axis represents the phase shift properties, while the y-axis displays the number of objects with a given electrical phase shift. The region from -π to -2 did not contain any relevant data and therefore the range has been set to -2 to 3.14. NB! The valley occurring at 1.57 (π/2), gives the impression that there are a total of three populations, but this dip is an Arctan artifact in the visual representation of data.  For more information see page 63 in this SBT PhD thesis. The lower plot shows the amplitude distribution. 

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Due to the presence of the two phase shift populations, it is not yet possible to unambiguously assign endospores to a single population. Curiously, the two populations appear to have different heat tolerance where the ~1.1 rad population is more tolerant to heat (85 °C, 15 min) than the other. In the subsequent plots, specific intervals (gating) for the high frequency (HF) channel have been applied for the two populations. We will later refer to these as impedance fingerprints:

• ~1.1 rad population: HFphase [0.8 : 1.3] + HFamplitude [-80 : -60]. Dark purple
• ~1.7 rad population: HFphase [1.3 : 2.2] + HFamplitude [-80 : -20]. Grey 

When the concentrations are inspected as a function of heat treatment time, the concentration of ~1.7 rad population (dark purple curve) is halved within 15 minutes, while the ~1.1 population (grey curve) shows a smaller drop in concentration over time. Knowing that endospores have remarkable heat tolerance it is more plausible that the spore population would be the ~1.1 rad population. This is further strengthened by the fact that the ~1.1 rad population shows good correlation to the colony forming units (CFUs) with a close to a 1:1 correlation for the different treatment times (bar chart). The concentration determined from the ~1.1 rad population is in good agreement with the 8x10⁶ to 5x10⁷ CFU/mL range provided by the BGA certificate of analysis, CoA, as indicated with the dashed box over the 0 min bars below.

In summary, from these data, the optimized impedance fingerprint for Bacillus subtilis BGA endospores are the HF custom gating parameters HFphase [0.8 : 1.3] and HFamplitude [-80 : -60]. These settings will be used throughout this application note.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

B. subtilis spore populations have different tolerance to heat: The x/y chart on the left shows that the population centered at ~1.7 rad has relatively poor heat tolerance, while the population centered at ~1.1 rad has high heat tolerance. The colony forming unit (CFU) concentrations are close to that of the ~1.1 rad population. This relationship is tested further in the bar chart on the right where the ratio between the ~1.1 rad population and the CFUs are relatively close to 100% for the different heat treatment times. The ~1.7 rad population follows a 2^nd order polynomial trend for reduction in number of objects as a function of heat treatment time (R² = 0.9884), while both the ~1.1 rad population and CFUs follow a linear trend as a function of heat treatment time (R² = 0.9312 and 0.8763, respectively). All analyses performed in triplicate dilution series. The CFU/mL accept range from the BGA certificate of analysis, CoA, is shown as a dashed box.  

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

After determining the custom gating for purified BGA spores we set out to investigate sporulation over time in more complex cultures of B. subtilis and B. thuringiensis. To ensure maximal production of spores, an optimized growth medium was kindly provided by Ohly GmbH. Full details of the medium can be accessed here. 100 mL sterile-filtered medium in baffled Erlenmeyer shake flasks were inoculated with a colony from an agar plate and incubated (35 C, 200 RPM). The B. subtilis strain was the same as for the initial purified spore experiments (DSM 618). Samples were withdrawn over time and subjected to bead beating with glass beads for 1 min at max speed (bead genie) followed by BactoBox analyses and microscopy on an Etaluma LS620 microscope equipped with a 60x long working distance phase contrast objective and three fluorescence channels. Three visualization principles were used for microscopic evaluation:

• Phase contrast: Vegetative cells will appear as black objects. Spores will appear as bright, refractive objects.,
• SYBR green I: This cyanine dye is permeant to membranes and will stain nucleic acids and emit light at 520nm, predominantly in the green fluorescence channel. Some bleed-through is observed in the blue channel. Here, the acquisition settings were set to exploit the green channel for detection of weakly fluorescent objects, e.g. the difficult-to-stain endospores and exploit the blue channel for visualization of vegetative cells.
• TOTO-3: This cell-impermeant thiazole red homodimer will stain nucleic acids in cells with compromised membranes and have maximum emission at 661nm in the red fluorescence channel.

After 10-20 hours, the cells are well within the exponential stage. For B. subtilis day 0 and day 1 phase contrast micrographs (upper left row) feature mainly large, dark rod-shaped single cells and chains with intense staining by SYBR and relatively poor staining with TOTO-3. These are likely vegetative cells devoid of pre-spores and endospores. On Day 2, bright, refractive, small objects have appeared within the rods with distinct staining of a TOTO-3-positive part and a part with less intense TOTO-3 staining. These objects are likely pre-spores and final spores. On day 7 mainly small, refractive endospores are observed with few rod-shaped objects present.

For B. thuringiensis, bright, refractive objects appear inside the large rod-shaped cells already on day 1. On day two, no rod-shaped cells are seen and the same goes for day 7, where only small objects are observed. Contrary to the B. subtilis endospores the B. thuringiensis endospores give relatively intense staining with SYBR. Also note that B. thuringiensis is more permeable to TOTO-3 during day 0 and 1 than B. subtilis indicating that prespore formation occurs very early for B. thuringiensis. In summary, the microscopic data indicates that B. thuringiensis forms endospores earlier than B. subtilis and that the sporulation appears more complete for B. thuringiensis with no detection of rod-shaped objects in the 7-day micrographs.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Microscopic progression from vegetative cells to endospores: Bacillus shake flasks assayed from the early vegetative cycle to the final sporulation stage. Each column represents the incubation time and each row represents the microscope channel for a fixed x/y position on the microscope slide. Upper row is the phase contrast image. SYBR-green emission mainly occurs in the green channel but also bleeds into the blue channel. The exposure settings for the green channel have been set to enhance detection of weakly fluorescent objects like endospores. The acquisition settings for the blue channel have been balanced for visualization of vegetative cells. The lowest row features fluorescence emission from TOTO-3 that mainly stains bacteria with non-intact membranes. A 5 µm scale-bar is shown in all images. Note that the samples on Day 7 have been concentrated x5 using centrifugal filters with 1,000,000 molar weight cutoff, MWCO.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

The microscopic inspection shows that B. subtilis is predominantly in a vegetative state at 15.5H and 31.5H. This is also the case when measured with BactoBox analyses in the upper left part of the figure with the intact cell range of the HF phase distributions (shown in violet). Two additional populations are appearing at 31.5H: a population with conductive objects centered at -2 rad (green part) and a population with poorly conductive properties (grey part). The objects with impedance signatures corresponding to the purified BGA spores mainly appear later than 31.5H (dark purple part).  

For B. thuringiensis, the vegetative cells are also mainly present at 15.5H and 31.5H, but curiously, the conductive objects (green part) are completely absent. The amplitude distributions (turquoise curves) indicate that some of the objects are extremely large, which is consistent with the huge (>20µm) chains in the phase contrast images at 15.5H (right part of the figure). Of note, objects larger than 5µm will typically not enter the measurement channel, and therefore improved homogenization of chainformers must be done before the quantitative results for B. thuringiensis at the early time points can be trusted. For B. subtilis, the events have lower amplitude, which means that the quantitative results are more reliable.   
The highly conductive objects at -2 rad are typically observed when (especially G+) bacteria have compromised membranes, but still have somewhat intact cell peptidoglycan cell walls. As mentioned in the introduction, B. thuringiensis needs to efficiently release toxins from the interior of the cell, and therefore the mother cell state with a permeabilized membrane is expected to be very short. This fits well with the absence of the conductive objects at -2 rad. For B. subtilis, the -2 rad population appears to be more persistent and somewhat variable over time. In principle, this population could harbor both dead cells (ghosts) as well as endospore-containing mother cells with non-intact membrane. For convenience, this population will be referred to as “mother cells + ghosts”.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

BactoBox tracks sporulation in real time: 3D-plot of the phase shift distributions (left), reflecting the electrical properties of Bacillus objects. Upper part represents B. subtilis while the lower part represents B. thuringiensis. X-axis depicts high frequency (HF) phase shift angle in radians, while the probability density function is shown on the y-axis. Z-axis reflects the incubation time. The impedance signatures underlying the grey, green, dark purple and violet coloring are described in the text. HF amplitude distribution is represented on a log scale from -80 to -20 dB. Small objects are seen at high negative values, while larger objects move right towards smaller negative values. The two phase contrast micrographs show the presence of either only vegetative cells (15.5 H) or mainly spores (101.5 H).

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Below, the B. subtilis data have been subjected to custom classification for the three observed populations when adjusted for dilution factor. Early in the growth curve, the vegetative cells dominate (violet curve) until 31.5 hours where this population drops in concentration. At this time point, two other populations increase in concentration, namely the “mother and ghost cells” (green curve) and the “poorly conductive” population just right of the spore population in the phase shift diagram (grey curve). After the 31.5 H sampling point, these populations drop in concentration to stabilize after ~49.4 H. The region corresponding to the bona fide purified spores (black curve) starts to appear when the intact cell concentration drops. The endospore concentration increases at least to the last sampling point. The pie charts in the upper part of the figure indicates the relative abundance of each species. The most striking change over time is the reduction of intact cells from virtually 100% of the detected events at 15.5 H to almost nothing after 101.5 H. Also, the spores with the narrow custom gating constitute an almost negligible part until after the 60.8 H sampling point.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Quantitative B. subtilis changes from vegetative to sporulation cycle monitored by BactoBox: The individual populations from the phase shift diagram adjusted for dilution factors and depicted as absolute concentrations. The pie charts show the relative concentrations of the three populations for each sampling point with respect to the incubation time. Open brackets in the legend for the custom gating indicates that the outside limits are used for classification, while closed brackets indicate inside limits. Note that the intact cell region has been narrowed slightly from the default ]-2.72:1.83[ limits based on comparison with qualitive results from fluorescence microscopy (results not shown). Empirical concentration correction (ECC) factors were not used in the present dataset.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

For the B. subtilis growth curves, the spore concentrations obtained by narrow gating constitutes a surprisingly small proportion (12%) of the total particles, while the proportion is higher for B. thuringiensis (18%). It is possible that some of the endospores are still contained within mother cells and that e.g. enzymatic or heat treatment is necessary to release them from the mother cell “cages”. The effect of heat treatment is investigated below (phase shift diagram, left part). Initially, before heat (BH), the 171H B. subtilis sample shows a complex distribution with the “mother and ghost cells”, “poorly conductive” and endospore populations (black curve). After heat treatment (AH), the dominant population is the ~1.1 rad endospore population (cyan). This could indicate that the heat treatment liberates a proportion of the endospores from the mother cells. It is also possible that some state of endospores are found in the poorly conductive region.

For B. thuringiensis, the populations are more similar before (green) and after (red) heat treatment, although some left-shifting of the populations is observed indicating that the objects become less electrically conductive after heat treatment.

When the endospore-gated populations are compared with plate counts, the AH measurements for B. subtilis are closer to plate counts than the BH measurements. For B. thuringiensis, the correlation is also improved, but not as dramatically as for B. subtilis. 

In summary, the B. thuringiensis endospores appear to be effectively released from the mother cells during sporulation while e.g. heat treatment is necessary to liberate the endospores from B. subtilis.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Heat treatment is needed to release B. subtilis but not B. thuringiensis endospores: Left phase shift diagram shows B. subtilis and B. thuringiensis endospores before and after heat treatment at 85 °C for 15 min. The black and green curves are before heat (BH) while the cyan and red curves are after heat treatment (AH). The white region indicates the default intact cell classification limits, while the grey region indicates other objects than intact bacterial cells. Right part of the figure shows the BactoBox and plate count results for the samples from the phase shift diagram. Plate counts were performed in triplicates plating from the same dilution series, while BactoBox measurements were performed in 1-plicates.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

When enumerating endospores, it is important to keep an eye on the background from other objects with poorly conductive properties. This is because the endospore phase shift is relatively close to that of e.g. microplastic particles released from the peristaltic tubing in the BactoBox. Two workarounds are possible:

1. Check the background from the system using a sterile-filtered “blank” diluent sample and subtract this from the result.
   
2. Perform measurement at relatively high spore concentrations where the background from the system is negligible, e.g. >1,000,000 total particles/mL.
   

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Streptomyces exospores are formed by a completely other mechanism than endospores. Upon starvation, the bacterial chains (filaments) grow into the air and desiccate to form exospores that can be spread to more nutrient-rich environments. Contrary to endospores, the exospores are wrapped in a single cytoplasmic membrane and the cytoplasmic water content is also comparable to normal vegetative bacteria. Therefore - in theory - the exospores should be more electrically conductive than the endospores. When subjected to BactoBox measurements, this indeed turns out to be the case (left part of the figure below). In fact, the exospores have phase shift properties very similar to normal vegetative bacteria. Moreover, with no need for custom gating, the bar chart shows that the exospore concentration determined by BactoBox is in very good agreement with plate counts.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Streptomyces griseus exospores have similar electrical properties as vegetative cells: Phase shift diagram of exospores scraped from a 7 day S. griseus DSM 40855 tryptic soy agar (TSA) plate with 5% sheep’s blood. Practically all objects are confined to the intact cell range (white region). The micrograph shows the green fluorescence channel from SYBR green-stained exospores revealing very homogeneous, small exospores (40x objective, 5 µm scalebar shown in yellow). The bar chart shows a head-to-head comparison of BactoBox intact cell concentrations (ICC) and plate counts, all performed in dilution series triplicates. 

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Endospores are tedious to enumerate with cultivation-based methods and FFC requires expensive analytical instruments and skilled operators. BactoBox represents a promising alternative to plate counts and the impedance signatures for endospores are clear cut: Firstly, the small size leads to low-amplitude populations and secondly, the presence of a double set of membranes results in distributions very close to the entirely non-conducting region of the phase shift diagram.

For purified spores, BactoBox shows good agreement with orthogonal plate counts results also after heat treatment. For complex, sporulating cultures, BactoBox gives real-time, simultaneous, qualitative information on four different populations: Vegetative (intact) cells, released endospores, mother + ghost cells, and objects with poorly conductive properties. Best of all, the BactoBox instrument is affordable, sample workup is fast, and the procedure is easy to standardize.

Here, BactoBox gave insights in the sporulation landscapes for B. subtilis and B. thuringiensis. Consistent with established findings, B. thuringiensis releases spores very efficiently from mother cells, likely concomitant with release of insecticide-like toxins. Endospore release from B. subtilis was conversely not as effective and the plausible mother/ghost cell population was present throughout the sporulation stage.

Finally, it was shown that BactoBox works well for exospores from Streptomyces with a close to 1:1 agreement with plate counts. Instead of the non-conductive properties of the endospores, the exospores were associated with the same phase shift properties as vegetative cells.

In summary the advantages of using BactoBox for spore enumeration are highlighted below:

• Fast: While plate count takes several days (or weeks) for all the spores to hatch, BactoBox provides the results within few minutes.
• Informative: Enumeration of both vegetative cells and spores.
• Precise: Coefficient of variation is typically within 5-10% for purified spores.
• Accurate: Close agreement with plate count results for purified spores 

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

The advantages above greatly accelerate the learning loops for optimizing spore production. With plate counts several days of incubation would be needed to enumerate vegetative cells and endospores. With BactoBox, you get the results in real time and therefore the subsequent optimization tests can be initiated immediately. For purification of spores, the impedance data can unveil if spores appear to be trapped in certain populations and assess the presence of unwanted particulate impurities. The typical use case is to track fermentation processes for spore-producing bacteria, e.g. to optimize the medium constitution or maximize the yield of the final spore product.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

To harness the power of BactoBox for spore enumeration, you should first establish the proper phase and amplitude limits for your specific species. This can be done easily by post-analysis in the data visualization tool, BactoBox Explorer. Subsequently, these settings can be uploaded to BactoBox as a custom program for real-time measurements. Contact [email protected] to get started with BactoBox Explorer.  
Inspiration for gating is provided in the table below. It is also possible to download the BactoBox classification files for Bacillus endospores and Streptomyces exospores by clicking here and unzipping the file. 

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Some questions remain: What is the most effective sample treatment to liberate endospores from mother cells. What does the “poorly conductive” region in the phase shift diagram correspond to. And where do the crystal protein toxins from B. thuringiensis occur in the phase shift diagrams. More studies and orthogonal techniques are needed to answer these questions. Also, there are other, interesting, sporulating microorganisms out there. This application note will be updated when BactoBox measurements are available on clostridial and fungal spores. We welcome you to join us in the quest for answers. By submitting (anonymized) data on growth curves from sporulating organisms it can be investigated if the impedance fingerprints for BGA spores are representative of all endospores or if the parameters must be optimized for the individual species. Please contact us at [email protected] if you are interested in contributing.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

• OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
• Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
• OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results. 
• Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.   
• Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
•  Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
• Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.