# README — Crystal violet biofilm assay: *Cupriavidus necator* H16 wild-type versus ΔB2043

## 1\. Formal information

* **Dataset title:** Crystal violet biofilm assay comparing *Cupriavidus necator* H16 wild-type and the ΔB2043 mutant
* **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:** 2026-01-15
* **Version:** 1.0
* **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; Markdown README

## 2\. Research context and objective

This dataset contains crystal violet assay measurements used to compare surface-associated biofilm biomass of *Cupriavidus necator* H16 and a markerless deletion mutant lacking B2043. B2043 encodes a GGDEF/EAL-domain protein implicated in c-di-GMP-dependent biofilm regulation. The assay was designed to determine whether deletion of B2043 alters biofilm-associated biomass after 72 h of static heterotrophic cultivation on fructose.

## 3\. Experimental methods

The strains were grown overnight in LB medium and the OD600 was measured. Cultures were adjusted to an OD600 of 0.15 in 200 µL modified minimal medium per well. The medium was DSMZ medium 81 containing 2.5 mg ferric ammonium citrate instead of the standard 50 mg and was supplemented with 20 mM fructose. Outer wells were filled with uninoculated minimal medium to reduce evaporation. The inoculated plate was incubated for 72 h at a setpoint of 30 °C under static conditions.

After incubation, OD600 was measured with a Tecan Infinite 200Pro. The medium was removed and the wells were washed three times with minimal medium. Adherent cells were stained with 300 µL of 0.1% crystal violet for 15 min. The stain was removed, and bound crystal violet was solubilized with 200 µL of 30% acetic acid for 10 min. Absorbance was then measured at 590 nm.

The biofilm index was calculated for each well as:

`biofilm\\\_index = A590 / OD600`

Each biofilm index was additionally normalized by dividing it by the mean biofilm index of the wild-type strain measured on the same plate:

`normalized\\\_biofilm\\\_index = biofilm\\\_index / mean\\\_biofilm\\\_index\\\_WT`

## 4\. Biological material and experimental design

* **Organism:** *Cupriavidus necator*
* **Wild type:** *C. necator* H16
* **Mutant:** *C. necator* H16 ΔB2043
* **Replicate type:** Biological replicates
* **Replicates present in the uploaded file:** 12 wells per strain
* **Wild-type wells:** B3, C3, D3, E3, B5, C5, D5, E5, C8, C9, E8, E9
* **ΔB2043 wells:** B2, C2, D2, E2, B4, C4, D4, E4, D8, D9, B8, B9

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

### Plate reader

* **Instrument:** Tecan Infinite 200Pro
* **Serial number:** 2106012005
* **Control software:** Tecan i-control, version 2.0.10.0
* **Firmware:** V\_5.31\_04/18\_InfiniteRX, dated 2018-03-28
* **Instrument purchase year:** 2021
* **Maintenance:** Regularly maintained;
* **Pre-measurement check:** Internal instrument self-tests were completed immediately before measurement
* **Plate:** Greiner 96-well flat-bottom transparent polystyrene plate;
* **Instrument shaking before measurement:** Orbital shaking for 20 s at 1 mm amplitude

### Measurement settings

* **OD600:** wavelength 600 nm; bandwidth 9 nm; 25 flashes; settle time 0 ms; recorded temperature 30.1 °C
* **Crystal violet absorbance:** wavelength 590 nm; bandwidth 9 nm; 25 flashes; settle time 0 ms; recorded temperature 28.0 °C
* **Incubation setpoint:** 30 °C
* **Recorded measurement-temperature range:** 28.0–30.1 °C

### Data curation software

* Python 3.13.5, using only the Python standard library, was used to restructure the original export into machine-readable CSV files and to recalculate descriptive statistics.
* Original spreadsheet used to calculate the reported p-value

## 6\. File structure

### `260115\\\_crystal\\\_violet\\\_assay\\\_wt\\\_vs\\\_deltaB2043.csv`

Cleaned long-format dataset with one row per measured biological replicate well.

|Column|Description|Unit / allowed values|
|-|-|-|
|`dataset\\\_id`|Unique identifier for this experiment|text|
|`experiment\\\_date`|Date of the assay|ISO 8601 date|
|`assay`|Experimental assay|`crystal\\\_violet\\\_biofilm\\\_assay`|
|`organism`|Organism name|text|
|`strain`|Full strain label|text|
|`genotype`|Machine-readable genotype|`wild\\\_type`, `delta\\\_B2043`|
|`replicate\\\_type`|Type of replicate|`biological`|
|`biological\\\_replicate`|Sequential replicate number within each strain|integer|
|`well`|96-well plate position|text|
|`medium`|Machine-readable medium description|text|
|`carbon\\\_source`|Carbon source|`fructose`|
|`carbon\\\_source\\\_concentration\\\_mM`|Fructose concentration|mM|
|`initial\\\_OD600`|OD600 used to inoculate each well|dimensionless absorbance|
|`culture\\\_volume\\\_uL`|Culture volume per well|µL|
|`incubation\\\_duration\\\_h`|Static incubation time|h|
|`incubation\\\_temperature\\\_setpoint\\\_C`|Incubation setpoint|°C|
|`incubation\\\_mode`|Incubation mode|`static`|
|`absorbance\\\_590\\\_nm`|Absorbance after CV staining and solubilization|dimensionless absorbance|
|`OD600\\\_after\\\_72h`|Culture OD600 after incubation|dimensionless absorbance|
|`biofilm\\\_index\\\_A590\\\_per\\\_OD600`|A590 divided by OD600|dimensionless ratio|
|`biofilm\\\_index\\\_normalized\\\_to\\\_WT\\\_mean`|Biofilm index divided by the WT mean on the same plate|dimensionless ratio|
|`blank\\\_correction\\\_status`|Documentation of reported blank correction|text|
|`instrument`|Plate-reader model|text|

## 7\. 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.