# README — OCT biovolume quantification of *Cupriavidus necator* H16 wild-type and ΔB2043

## 1\. Formal information

* **Dataset title:** OCT-based biovolume quantification of *Cupriavidus necator* H16 wild type and ΔB2043 biofilms in a flow-through system
* **Dataset DOI:** 10.15480/882.17412
* **Dataset creators:**

  * Janek R. Weiler, ORCID: 0000-0003-4930-5764, E-mail: janek.weiler@tuhh.de, Affiliation: Institute of Technical Microbiology, Hamburg University of Technology, Hamburg, Germany
  * Miriam Edel-Teichmann, ORCID: 0009-0008-6283-0006, E-mail: miriam.edel@tuhh.de, Affiliation: Institute of Technical Microbiology, Hamburg University of Technology, Hamburg, Germany
* **Experiment date:** 2026-02
* **Dataset preparation date:** 2026-07-02
* **Version:** 1.1
* **Related publication:** Identification of genetic determinants that promote biofilm growth under heterotrophic conditions in Cupriavidus necator using transposon enrichment, DOI: \[TO BE COMPLETED]
* **Language:** English
* **Data formats:** UTF-8 CSV with comma delimiters and decimal points; Microsoft Excel XLSX; Markdown README

## 2\. Research context and objective

This dataset contains time-resolved optical coherence tomography (OCT) measurements used to compare biofilm development of *Cupriavidus necator* H16 and the ΔB2043 deletion mutant in a continuous-flow microfluidic cultivation system. OCT enabled non-invasive analysis of biofilm growth and three-dimensional structure. Biofilm development was quantified as biovolume per surface area.

## 3\. Microfluidic flow-cell cultivation

The cultivation system was based on the microfluidic setup described by Hansen et al. (2019; 10.1038/s41598-019-45414-6). Flow cells were constructed from polydimethylsiloxane (PDMS) using the Sylgard® 184 Silicone Elastomer Kit. PDMS was cast in a brass negative mold and cured at 60 °C for 2 h. Placeholder cannulas created separate ports for medium inflow, medium outflow, and inoculation.

The PDMS body was covalently bonded by oxygen-plasma treatment to 1-mm-thick cover glass. Permeable cannulas with a diameter of 0.8 mm were inserted into the ports and connected to silicone tubing with a 1.5 mm inner diameter. Medium delivery was controlled using Reglo ICC peristaltic pumps. Luer connectors and three-way valves enabled switching between inoculation and medium-supply modes. Inlet and outlet vessels were pressure-equilibrated using sterile 0.2-µm filters.

All compatible components were sterilized by autoclaving at 121 °C and 1 bar overpressure for 20 min. Final assembly was performed in a Thermo Scientific MSC Advantage Safety Workbench.

### Flow-cell geometry

* **Channel length:** 54 mm
* **Channel width:** 3 mm
* **Channel height:** 2 mm
* **Channel volume:** 324 µL
* **Channel volume including transition regions:** 354 µL

### Cultivation and inoculation conditions

* **Cultivation medium:** MM 81
* **Cultivation temperature:** 30 °C
* **Medium flow rate during cultivation:** 4 mL/h
* **Pre-culture adjustment:** OD600 = 0.2
* **Inoculation duration:** 2 h
* **Inoculation route:** side port
* **Inoculum flow rate during inoculation:** 1 mL/h
* **Medium flow rate during inoculation:** 3 mL/h
* **Measurement position:** middle section of the cultivation channel
* **Replicates:** three biological replicates per strain

## 4\. OCT acquisition and image analysis

OCT images were acquired with a GANYMEDE system equipped with an LSM03-BB objective lens (Gan210-SP4, Thorlabs, Dachau, Germany). The system used an infrared light source with a central wavelength of 930 nm and a bandwidth of 100 nm.Image acquisition and analysis were performed using ThorImage version 5.1.1 and

Fiji/ImageJ version 1.53/2.1.0. Data was analyzed according to Wagner and Horn (2017; 10.1002/BIT.26283).

The source workbook contains:

* measurement time in hours,
* replicate identifier,
* strain,
* segmented white-pixel count,
* calculated biovolume in µL/cm².

Measurements span 0–80 h in 4 h intervals.

## 5\. Biological material and experimental design

* **Organism:** *Cupriavidus necator*
* **Wild-type:** *C. necator* H16
* **Mutant:** *C. necator* H16 ΔB2043
* **Replicate type:** biological replicates
* **Number of replicates:** three per strain
* **Source replicate identifiers:** 1–3 for ΔB2043 and 4–6 for wild-type
* **Standardized replicate identifiers:** 1–3 within each strain
* **Time points:** 0, 4, 8, …, 80 h
* **Total observations:** 126

## 6\. Instrumentation, software, and quality control

* **OCT instrument:** GANYMEDE system, Gan210-SP4, Thorlabs, Dachau, Germany
* **Objective:** LSM03-BB, Thorlabs
* **Central wavelength:** 930 nm
* **Bandwidth:** 100 nm
* **Acquisition software:** ThorImage version 5.1.1
* **Image-analysis software:** Fiji; ImageJ version 1.53/2.1.0

## 7\. File structure

### `oct\_biovolume\_B2043\_vs\_wildtype.csv`

Cleaned long-format data with one row per biological replicate and time point.

|Column|Description|Unit / allowed values|
|-|-|-|
|`dataset\_id`|Dataset identifier|text|
|`experiment\_date`|Experiment date; not yet supplied|ISO 8601 date or empty|
|`assay`|Analytical method|text|
|`organism`|Organism name|text|
|`strain`|Full strain label|text|
|`genotype`|Machine-readable genotype|`wild\_type`, `delta\_B2043`|
|`replicate\_type`|Replicate classification|`biological`|
|`biological\_replicate`|Replicate number standardized within each strain|integer 1–3|
|`source\_replicate\_id`|Identifier retained from the source workbook|integer 1–6|
|`time\_h`|Time after the start of the measurement series|h|
|`white\_pixel\_count`|Number of segmented white pixels|pixels|
|`biovolume\_uL\_per\_cm2`|Calculated biofilm volume per surface area|µL/cm²|
|`cultivation\_system`|Type of flow-cell system|text|
|`flow\_cell\_material`|Flow-cell construction material|text|
|`flow\_cell\_length\_mm`|Channel length|mm|
|`flow\_cell\_width\_mm`|Channel width|mm|
|`flow\_cell\_height\_mm`|Channel height|mm|
|`reported\_flow\_cell\_volume\_uL`|Flow-cell volume stated in the methods|µL|
||||
|`cover\_glass\_thickness\_mm`|Cover-glass thickness|mm|
|`tubing\_inner\_diameter\_mm`|Inner diameter of silicone tubing|mm|
|`cultivation\_medium`|Medium used during flow cultivation|text|
|`cultivation\_temperature\_C`|Cultivation temperature|°C|
|`cultivation\_medium\_flow\_rate\_mL\_per\_h`|Medium flow rate after inoculation|mL/h|
|`inoculation\_duration\_h`|Duration of inoculation|h|
|`inoculum\_flow\_rate\_mL\_per\_h`|Inoculum flow through the side port|mL/h|
|`medium\_flow\_rate\_during\_inoculation\_mL\_per\_h`|Medium flow during inoculation|mL/h|
|`inoculum\_initial\_OD600`|OD600 of the adjusted pre-culture|dimensionless|
|`measurement\_location`|Position in the channel used for OCT analysis|text|
|`instrument`|OCT system|text|
|`objective\_lens`|Objective lens|text|
|`center\_wavelength\_nm`|Central wavelength|nm|
|`bandwidth\_nm`|Spectral bandwidth|nm|
|`acquisition\_software`|Image-acquisition software|text|
|`image\_analysis\_software`|Image-analysis software|text|
|`analysis\_method\_reference`|Reference to image-analysis method|text|

## 8\. Data-processing and quality notes

1. All 126 observations were retained without exclusion or replacement.
2. Strain labels were standardized to `wild\_type` and `delta\_B2043`.
3. Replicate identifiers were standardized to 1–3 within each strain while retaining the original identifiers.
4. The source heading `white pixcels` was corrected to `white\_pixel\_count`.
5. Biovolume values stored as text were converted to numeric decimal-point notation without changing the values.
6. The relationship between white-pixel count and biovolume corresponds to an average conversion factor of approximately `1.5845273331e-07` µL cm⁻² per white pixel (range `1.5838522295e-07`–`1.5851400521e-07`).
7. No outlier removal was performed.

## 9\. Rights, access, and reuse

* **Access restrictions:** None
* **License:** Public Domain Mark 1.0 Universal
* **Personal or sensitive data:** None
* **Preferred citation:**

	`Weiler, JR et al. (2026). Crystal violet biofilm assay comparing Cupriavidus necator H16 wild type and the ΔB2043 mutant. Version 1.0. TORE. https://doi.org/10.15480/882.17412`

	When reusing the dataset, users should cite both the dataset DOI and the associated publication DOI once available.